Pyrrolopyrimidine nucleosides and analogs thereof

ABSTRACT

The present disclosure provides pyrrolopyrimidine nucleoside analogs of the Formula I, Formula IA, Formula IB, or Formula II and phospholipid conjugates and pharmaceutical compositions thereof wherein R c  and A are defined herein. Also presented are methods of treating and/or preventing viral infection and/or viral infection-associated disease or disorder with one or more compounds of Formula I, Formula IA, Formula IB, or Formula II.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/231,528, filedAug. 8, 2016, which claims priority to and the benefit of U.S.Provisional Application No. 62/202,010, filed Aug. 6, 2015, and to U.K.Application No. 1606645.8, filed Apr. 15, 2016, the contents of whichare incorporated by reference herein in their entirety.

GOVERNMENT INTEREST

This invention was made with government suport under Grant No. 5U19-AI-031718 (National Cooperative Drug Discovery Group forOpportunistic Infections: New Inhibitors and New Targets to Develop HCMVDrugs), awarded by the National Institute of Allergy and InfectiousDiseases. The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file named “CHIM-830_C01US_ST25.txt”, which wascreated on Jan. 30, 2017 and is 2.18 KB in size, are hereby incorporatedby reference in their entireties.

TECHNICAL FIELD

This application relates to pyrrolopyrimidine nucleoside analogs andphospholipid conjugates thereof and methods of synthesis thereof. Thepyrrolopyrimidine nucleoside analogs and their phospholipid conjugatescan be used as antiviral agents for treating viral infections. Thisapplication also relates to pharmaceutical compositions comprisingpyrrolopyrimidine nucleoside analogs and phospholipid conjugatesthereof.

BACKGROUND

Viral infections can have serious adverse effects on individuals andsociety as a whole. In addition to fatal viral infections such as ebola,even non-fatal infections can have serious societal and economicconsequences. For example, human noroviruses (NV) are the most commoncause of epidemic acute gastroenteritis worldwide with an estimated19-21 million cases each year in the United States including56,000-71,000 hospitalizations and 570-800 deaths (Hall et al., Emerg.Infect. Dis. 2013 August; 19(8):1198-205).

Accordingly, development of an effective antiviral treatment effectiveagainst viruses is important to improve the health of infectedindividuals and as a public health measure to prevent outbreaks of otherpathogenic viruses.

SUMMARY

The present disclosure provides pyrrolopyrimidine nucleoside analogs andphospholipid conjugates thereof. Also included are pharmaceuticalcompositions comprising the same and methods of synthesis thereof.

The present disclosure also provides methods of treating and/orpreventing viral infection and/or viral infection-associated disease ordisorder with one or more compounds of the present embodiments. Thedisclosure addresses the need for new therapies that can be used totreat and/or prevent viral-induced disease using novel antivirals anddelivery vehicles.

In one aspect, the present disclosure relates to compounds of Formula I:

and pharmaceutically acceptable salts, solvates, enantiomers,diastereomers, racemates and mixtures thereof, wherein:

A is:

X₁ is CR₁₁R₁₂ or OCH₂CH₂ with the oxygen atom distal to the R^(I) moietyin A, in which R₁₁ and R₁₂ are independently hydrogen or substituted orunsubstituted C₁-C₄ alkyl;

X₂ is absent, —O—, —C(O)O—, or —OCH₂— with the oxygen atom distal to theR^(I) moiety in A;

each R^(I) independently is hydrogen, substituted or unsubstituted C₁-C₆alkyl,

or R^(I) is an amino acid residue bound via the carbonyl group of X₂;

v is 0 or 1;

n is 0, 1, 2, or 3 and when X₂ is —C(O)O—, n is 0;

-   -   p is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11;    -   q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        or 18;

R_(z) is hydrogen, halogen, C₁-C₄ alkylthio, C₁-C₄ alkoxy, substitutedor unsubstituted C₁-C₄ alkyl, substituted or unsubstituted C₂-C₄alkenyl, substituted or unsubstituted C₂-C₄ alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl;or substituted or unsubstituted non-aromatic heterocyclic ring;

R_(a), R_(b), R_(x), and R_(y) are each independently selected from thegroup consisting of hydrogen, halogen, OH, SH, substituted orunsubstituted C₁-C₆ alkoxy, substituted or unsubstituted aryloxy,substituted or unsubstituted C₁-C₆ alkylthio, substituted orunsubstituted arylthio, substituted or unsubstituted —O-carbonylalkyl,substituted or unsubstituted —O— carbonylaryl, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₂-C₆ alkenyl,substituted or unsubstituted C₂-C₆ alkynyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted cycloalkyl, and substituted or unsubstituted cycloaklenyl;

alternatively any R_(a) or R_(b) in (CR_(a)R_(b))_(p) is taken withanother R_(a) or R_(b), together with the atoms to which they areattached and any intervening atoms therebetween to form a carbon-carbondouble or triple bond, a C₆-C₁₀ aryl, 5- to 10-membered heteroaryl,C₃-C₁₀ cycloalkyl, C₄-C₁₀ cycloalkenyl, or 5- to 10-memberednon-aromatic heterocyclic ring structure; or any R_(x) or R_(y) in(CR_(x)R_(y))_(q) is taken with another R_(x) or R_(y), together withthe atoms to which they are attached and any intervening atomstherebetween to form a carbon-carbon double or triple bond, a C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀ cycloalkyl, C₄-C₁₀cycloalkenyl, or 5- to 10-membered non-aromatic heterocyclic ringstructure; or any CR_(a)R_(b) or CR_(x)R_(y) is replaced by oxygen,sulfur, sulfinyl (SO) or sulfonyl (SO₂);

R₁ and R₄₅ are each independently hydrogen, halogen, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₃-C₆cycloalkyl, substituted or unsubstituted C₂-C₆ alkenyl, substituted orunsubstituted C₄-C₈ cycloalkenyl, substituted or unsubstituted C₂-C₆alkynyl, substituted or unsubstituted C₈-C₁₂ cycloalkynyl, azido, —OH,substituted or unsubstituted C₁-C₆ alkoxy, substituted or unsubstitutedamino, —SH, or substituted or unsubstituted C₁-C₆ alkylthio;

each of R₂, R₃, R₄ and R₄₄ independently is hydrogen, halogen,substituted or unsubstituted C₁-C₆ alkyl, N₃, OH, substituted orunsubstituted C₁-C₆ alkoxy, substituted or unsubstituted amino, SH, orsubstituted or unsubstituted C₁-C₆ alkylthio; alternatively R₃ and oneof R₄ and R₄₄ together with the atoms to which they are attached form acarbon-carbon double bond;

R₅ is hydrogen, R^(I), M⁺, substituted or unsubstituted aryl,substituted or unsubstituted aralkyl, substituted or unsubstituted C₁-C₆alkyl, substituted or unsubstituted C₁-C₆ heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted non-aromaticheterocyclic ring, or substituted or unsubstituted heteroaryl; whereinM⁺ is a cation and wherein R₅ is not an amino acid; and

R_(c) is substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted C₃-C₆ cycloalkyl, substituted or unsubstituted C₂-C₆alkenyl, substituted or unsubstituted C₄-C₈ cycloalkenyl, substituted orunsubstituted C₂-C₆ alkynyl, substituted or unsubstituted C₈-C₁₂cycloalkynyl, or substituted or unsubstituted aryl.

In another aspect the present disclosure relates to compounds of FormulaIA:

and pharmaceutically acceptable salts, solvates, enantiomers,diastereomers, racemates or mixtures thereof, wherein:

A is:

X₁ is —CR₁₁R₁₂— or —OCH₂CH₂— wherein the oxygen atom is distal to theR^(IA) moiety in A;

R₁₁ and R₁₂ are independently hydrogen or C₁-C₄ alkyl, wherein the alkylis optionally substituted with one or more halogen, —OH, —SH, or —NH₂;

X₂ is absent, —O—, —C(O)O—, or —OCH₂— wherein the oxygen atom is distalto the R^(IA) moiety in A;

X₃ is independently —O— or —NH—;

B is independently —C(O)NH₂, aryl, or heteroaryl;

C is independently —OR, —NHR, or —N═CHN(R)₂;

each R^(IA) is independently is hydrogen or —C₁-C₆ alkyl, wherein thealkyl is optionally substituted with one or more —OH, —SH, or —NH₂, oxo,R_(a), or —OR_(a);

or R^(IA), is an amino acid residue bound via the carbonyl group,

v is 0 or 1;

n is 0, 1, 2, or 3 and when X₂ is —C(O)O—, n is 0;

-   -   p is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11;    -   q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        or 18;

R_(z) is hydrogen, halogen, —C₁-C₄ alkylthio, —C₁-C₄ alkoxy, —C₁-C₄alkyl, —C₂-C₄ alkenyl, —C₂-C₄alkynyl, aryl, heteroaryl, —C₃-C₈cycloalkyl, —C₄-C₈ cycloalkenyl, or 3- to 5-membered nonaromaticheterocycle, wherein each alkylthio, alkoxy, alkyl, alkenyl, alkynyl,aryl, heteroaryl, cycloalkyl, cycloalkenyl, or heterocycle is optionallysubstituted with one or more halogen, —OH, —SH, or —NH₂;

R_(a), R_(b), R_(x), and R_(y) are each independently selected from thegroup consisting of hydrogen, halogen, —OH, —SH, —C₁-C₆ alkoxy, aryloxy,—C₁-C₆ alkylthio, arylthio, —OC(O)C₁-C₆ alkyl, —OC(O)aryl, —C₁-C₆ alkyl,—C₂-C₆ alkenyl, —C₂-C₆ alkynyl, aryl, heteroaryl, —C₃-C₈ cycloalkyl, and—C₄-C₈ cycloaklenyl, wherein each alkoxy, aryloxy, alkylthio, arylthio,alkyl, aryl, alkenyl, alkynyl, heteroaryl, cycloalkyl, or cycloalkenylis optionally substituted with one or more halogen, —OR₁₁, —SR₁₁, or—NR₁₁R₁₂;

or any two R_(a) or R_(b), together with the atom to which they are bothattached, can combine to form a C₃-C₈ spirocycloalkyl or 3- to8-membered spiroheterocycle;

or any two R_(a) or R_(b), when on adjacent atoms, can combine to form acis- or trans-carbon-carbon double bond or a carbon-carbon triple bond;

or any two R_(a) or R_(b), when on adjacent atoms, can combine to forman oxo, aryl, heteroaryl, —C₃-C₁₀cycloalkyl, —C₄-C₁₀cycloalkenyl, or 5-to 10-membered ring heterocycle;

or any CR_(a)R_(b) can be replaced by —O—, —S—, —S(O)—, or —SO₂—;

or any two R_(x) or R_(y), together with the atom to which they are bothattached, can combine to form a —C₃-C₈ spirocycloalkyl or 3- to8-membered spiroheterocycle;

or any two R_(x) or R_(y), when on adjacent atoms, can combine to form acis- or trans-carbon-carbon double bond or a carbon-carbon triple bond;

or any two R_(x) or R_(y), when on adjacent atoms, can combine to forman oxo, aryl, heteroaryl, —C₃-C₁₀cycloalkyl, —C₄-C₁₀cycloalkenyl, or 5-to 10-membered ring heterocycle;

or any CR_(x)R_(y) can be replaced by —O—, —S—, —S(O)—, or —SO₂—;

R₁ and R₄₅ are each independently hydrogen, halogen, —N₃, —OH, —NH₂,—SH, —C₁-C₆ alkyl, —C₃-C₆ cycloalkyl, —C₂-C₆ alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆ alkynyl, —C₈-C₁₂ cycloalkynyl, —C₁-C₆ alkoxy, or—C₁-C₆ alkylthio wherein each alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, alkoxy or alkylthio is independently substitutedwith one or more halogen, —N₃, —OH, —NH₂, or —SH;

R₂, R₃, R₄ and R₄₄ are each independently hydrogen, halogen, —N₃, —OH,—NH₂, —SH, C₁-C₆ alkoxy, or —C₁-C₆ alkylthio, wherein each alkyl,alkoxy, or alkylthio is optionally substituted with one or more halogen,oxo, —N₃, —OH, —NH₂, or —SH;

or R₃ and one of R₄ and R₄₄, together with the atoms to which they areattached, can form a carbon-carbon double bond;

or R₃ and one of R₄ and R₄₄, together with the atoms to which they areattached, can combine to form a 4- to 8-membered cycloalkyl orheterocycle optionally substituted with C₁-C₆ alkyl;

R₅ is independently hydrogen, —R^(IA), M⁺, aryl, aralkyl, —C₁-C₆ alkyl,—C₁-C₆ heteroalkyl, cycloalkyl, non-aromatic heterocyclic ring, orheteroaryl, wherein M⁺ is a cation and wherein each aryl, aralkyl,alkyl, heteroalkyl, cycloalkyl, heterocycle, or heteroaryl is optionallysubstituted with one or more halogen, —N₃, —OH, —NH₂, or —SH, andwherein R₅ is not an amino acid; and

R₁₁ and R₁₂ are each independently, at each occurrence, hydrogen,halogen, —OH, —SH, —C₁-C₆ alkoxy, aryloxy, arylthio, —OC(O)C₁-C₆ alkyl,—OC(O)aryl, —C₁-C₆ alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, aryl,heteroaryl, —C₃-C₈ cycloalkyl, and —C₄-C₈ cycloaklenyl, wherein eachalkyl, aryl, alkenyl, alkynyl, heteroaryl, cycloalky and cycloalkenyl isoptionally substituted with one or more halogen, —N₃, —OH, —NH₂, or —SH;

R_(c) is —C₁-C₆ alkyl, —C₃-C₆ cycloalkyl, —C₂-C₆ alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆ alkynyl, —C₈-C₁₂ cycloalkynyl, or aryl, whereineach alkyl, cycloalkyl, alkenyl, cycloalkenyl, or aryl is optionallysubstituted with one or more halogen, —N₃, —OH, —NH₂, or —SH; whereinany of the nitrogen atoms in the fused pyrimidine ring can be oxidized.

In another aspect the present disclosure relates to compounds of FormulaII:

and pharmaceutically acceptable salts, solvates, enantiomers,diastereomers, racemates or mixtures thereof, wherein:

Y is —C(O)—, or

wherein X¹ is independently O, NH, or S, X² is independently a bond,—O—, —S—, or —NH—, and X³ is independently —OR, —NHR_(II), or —SR^(II);

each R^(II) is independently —H, —C₁-C₂₀alkyl, —C₂-C₂₀alkenyl,—C₂-C₂₀alkynyl, —C₃-C₈ cycloalkyl, —C₄-C₈ cycloalkenyl, aryl,heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl isoptionally substituted with one or more halogen, oxo, R¹, —OR¹, —NR¹R²,—SR¹, —OC(O)R¹, —C(O)OR¹, —NHC(O)OR¹, or —NHC(O)R¹;

R^(a) and R^(b) are each independently, at each occurrence, —H,—C₁-C₂₀alkyl, —C₂-C₂₀alkenyl, —C₂-C₂₀ alkynyl, —C₃-C₈ cycloalkyl, —C₄-C₈cycloalkenyl, aryl, heteroaryl, or heterocyclyl, wherein each alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, orheterocyclyl is optionally substituted with one or more halogen, oxo—OR¹, —NR¹R², —SR¹, —OC(O)R¹, —C(O)OR¹—NHC(O)OR¹, or —NHC(O)R¹;

R¹ and R² are each independently, at each occurrence, —H, —C₁-C₂₀alkyl,—C₂-C₂₀alkenyl, —C₂-C₂₀alkynyl, —C₃-C₈ cycloalkyl, —C₄-C₈ cycloalkenyl,aryl, heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl isoptionally substituted with one or more halogen, oxo, —R³, —R⁴, —OR³,—NR³R⁴, —SR³, —OC(O)R³, —C(O)OR³, —NHC(O)OR³, or —NHC(O)R³;

R³ and R⁴ are each independently, at each occurrence, —H, —C₁-C₂₀alkyl,—C₂-C₂₀alkenyl, —C₂-C₂₀alkynyl, —C₃-C₈ cycloalkyl, —C₄-C₈ cycloalkenyl,aryl, heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl isoptionally substituted with one or more halogen, oxo, aryl, heteroaryl,—OH, —NH₂, —SH, —OC(O)H, —C(O)OH, —NHC(O)OH, or —NHC(O)H;

R^(d) is independently —H or -D; and

n is independently 0, 1, 2 or 3.

In another aspect, the present disclosure relates to a pharmaceuticalcomposition comprising a compound of Formula I, Formula IA, Formula IB,or Formula II, or a pharmaceutically acceptable salt, solvate,enantiomer, diastereomer, racemate or mixture thereof, and apharmaceutically acceptable carrier. In some embodiments the presentdisclosure relates to a pharmaceutical composition comprising compound1, or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof, and a pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutical compositioncan be used to treat a viral infection (e.g., norovirus).

In another aspect, the present disclosure provides a method of treatinga viral infection or a viral-infection associated disease or disorder,wherein the method comprises administering to a subject in need thereofan effective amount of a compound described herein (e.g., a compound ofFormula I, Formula IA, Formula IB, or Formula II), or a pharmaceuticallyacceptable salt, solvate, enantiomer, diastereomer, racemate or mixturethereof. In some embodiments the compound is compound 1, or apharmaceutically acceptable salt, solvate, enantiomer, diastereomer,racemate or mixture thereof. In some embodiments, the virus isnorovirus.

In another aspect, the present disclosure also relates to apharmaceutical formulation of the compounds disclosed herein, or apharmaceutically acceptable salt, solvate, enantiomer, diastereomer,racemate or mixture thereof, for use in a method for treating orpreventing a viral infection or viral infection associated disease ordisorder, e.g., a double stranded DNA (dsDNA) or a single stranded RNA(ssRNA) viral infection. In some embodiments the compound is compound 1.In some embodiments, the virus is norovirus.

In another aspect, the present disclosure also relates to use of acompound or a pharmaceutical formulation disclosed herein, or apharmaceutically acceptable salt, solvate, enantiomer, diastereomer,racemate or mixture thereof, in the manufacture of a medicament fortreating or preventing a viral infection and/or viral infectionassociated disease or disorder, e.g., an ssRNA viral infection. Thepharmaceutical formulation can comprise a compound of Formula I, FormulaIA, Formula IB, or Formula II, or a pharmaceutically acceptable salt,solvate, enantiomer, diastereomer, racemate or mixture thereof. In someembodiments the compound is compound 1, or a pharmaceutically acceptablesalt, solvate, enantiomer, diastereomer, racemate or mixture thereof. Insome embodiments, the virus is norovirus.

The present disclosure also relates to methods for treating orpreventing a viral infection and/or viral infection associated diseaseor disorder, e.g., an ssRNA viral infection. The method can compriseadministering to a subject in need thereof a compound of Formula I,Formula IA, Formula IB, or Formula II. In some embodiments the compoundis compound 1. In some embodiments, the virus is norovirus.

The present disclosure also relates to a compound described herein, or apharmaceutically acceptable salt, solvate, enantiomer, diastereomer,racemate or mixture thereof, for use in treating or preventing a viralinfection or a viral infection-associated disease or disorder. Thecompound can be a compound of Formula I, Formula IA, Formula IB, orFormula II. In some embodiments the compound is compound 1, or apharmaceutically acceptable salt, solvate, enantiomer, diastereomer,racemate or mixture thereof. In some embodiments, the virus isnorovirus.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. In the case of conflict, thepresent specification, including definitions, will control. In thespecification, the singular forms also include the plural unless thecontext clearly dictates otherwise. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference. The references cited herein are not admitted to be prior artto the claimed disclosure. In addition, the materials, methods, andexamples are illustrative only and are not intended to be limiting.

Other features and advantages of the present disclosure will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows murine norovirus titer (plaque forming units per mL) intissue and feces harvested 3 days post-infection as part of Study No. 1.

FIG. 1B shows murine norovirus titer (plaque forming units per mg oftissue) in tissue and feces harvested 3 days post-infection as part ofStudy No. 1.

FIG. 2A shows murine norovirus titer (plaque forming units per mL) intissue and feces harvested 3 days post-infection as part of Study No. 2.

FIG. 2B shows murine norovirus titer (plaque forming units per mg oftissue) in tissue and feces harvested 3 days post-infection as part ofStudy No. 2.

FIG. 3A shows the number of plaque forming units per gram of cecum fromStudy 1 on a linear scale.

FIG. 3B shows the number of plaque forming units per gram of feces fromStudy 1 on a linear scale.

FIG. 4 shows the first duplicate of the results demonstrating the invitro efficacy of compound 1 for inhibiting human norovirus compared to2′-C-methylcytidine triphosphate and compound 2.

FIG. 5 shows the second duplicate of the results demonstrating the invitro efficacy of compound 1 for inhibiting human norovirus compared to2′-C-methylcytidine triphosphate and compound 2.

FIG. 6 shows an overlay of the results of the first and second duplicateof the results demonstrating the in vitro efficacy of compound 1 forinhibiting human norovirus compared to 2′-C-methylcytidine triphosphateand compound 2.

FIG. 7a shows an HPLC plot of compound 1.

FIG. 7b shows an HPLC plot of compound 1 after slurrying 3 hours at roomtemperature.

FIG. 7c shows an HPLC plot of compound 1 after slurrying 3 hours at 50°C.

FIG. 7d shows an HPLC plot of compound 1 after slurrying 24 hours atabout room temperature.

FIG. 8a shows a ¹HMNR spectrum of compound 1 from about −2 to about 14ppm.

FIG. 8b shows a ¹HMNR spectrum of compound 1 from about 2 to about 9ppm.

FIG. 8c shows a ¹HMNR spectrum of compound 1 from about 0 to about 9ppm.

DETAILED DESCRIPTION

Nucleoside phosphonates (e.g., ribonucleoside derivatives) represent atarget class of antivirals to inhibit viruses which rely on viralencoded enzymes using ribonucleotides or deoxyribonucleotides assubstrates, such as certain viral polymerases for many RNA virusesand/or viral helicases for RNA (e.g., ssRNA) or DNA viruses. However,without wishing to be bound by theory, one block to efficacy for thisclass of antivirals is the requirement for biochemical modification ofthe administered agent inside target cells to form the active antiviralnucleoside triphosphate. In some embodiments, if a nucleoside isdelivered, three phosphorylation steps are required to form thetriphosphate. Delivery of nucleoside phosphonates effectively bypassesthe first phosphorylation, but can exacerbate problems of deliveringclinically useful amounts of the charged drug across the lipid bilayerssurrounding cells.

Without wishing to be bound by theory, lipid conjugation can be used todisguise oral drugs, including nucleoside phosphonates, as naturalcompounds that are readily absorbed by the body. Specifically, in someembodiments, nucleoside phosphonates can be modified to resemblepartially metabolized (monoacyl) phospholipids. In some embodiments, incontrast to normal diacylphospholipids, monoacyl lipid-modifiednucleosides can readily penetrate the enterocytes lining the lumen ofthe gut, enter the circulating blood and/or lymph and, unlike standarddrugs, remain intact. Consequently, the lipid moiety can do more thandeliver the nucleoside to the plasma; it can facilitate efficient uptakeinto the target cells. The lipid can be cleaved in the cytoplasmiccompartment of the target cells and in the case of nucleoside analogconjugates, can yield the corresponding monophosphate. Overall, thisstrategy can lead to greatly increased levels of the active antiviral atthe site of viral replication.

The present disclosure provides compounds, pharmaceutical compositions,and methods of synthesizing and using the compounds for treating orpreventing a viral infection or viral infection associated disease ordisorder, e.g., an ssRNA viral infection.

In some embodiments, the compounds of the present disclosure haveimproved efficacy/toxicity ratio compared to compounds in the art usedsimilarly.

DEFINITIONS

Certain compounds of the present disclosure and definitions of specificfunctional groups are also described in more detail below.

It will be appreciated that the compounds, as described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas disclosedherein, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure may be substituted with more thanone substituent selected from a specified group, the substituent may beeither the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. For purposes of this disclosure, heteroatoms such as nitrogenmay have hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms. The nitrogen and sulfur heteroatoms may optionally beoxidized, and the nitrogen heteroatom may optionally be quaternized.Examples of substituents on the moieties disclosed herein (e.g., alkyl,alkenyl, alkynyl, alkoxy, aryl, heteroaryl, cycloalkyl, cycloalkenyl,non-aromatic heterocycle groups) include, but are not limited to,alkenyl, alkynyl, halogen, haloalkyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, heteroaryl, aryl, cycloalkyl, cycloalkenyl, non-aromaticheterocycle, hydroxyl, carbamoyl, oxo, amino, nitro, azido, —SH, and—CN.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. In particular one,some, or all hydrogens may be deuterium. Radioactive isotopes may beused, for instance for structural analysis or to facilitate tracing thefate of the compounds or their metabolic products after administration.By way of general example and without limitation, isotopes of hydrogeninclude deuterium and tritium and isotopes of carbon include C-13 andC-14. For example, compounds of Formula I include those wherein R₁ is Hor D; R₂ and R₃ are independently H, D, OH, OD, CH₃, or CD₃; and/or R₄is H, D, CH₃, or CD₃.

The term “independently” is used herein to indicate that the variable,such as atom or functional group, which is independently applied, variesindependently from application to application. For example, where morethan one substituent or atom (carbon or heteroatom, such as oxygen (O),sulfur (S), or nitrogen (N)) occurs, each substituent or atom isindependent of another substituent or atom and such substituents or atomcan also alternate.

The term “alkyl”, as used herein, refers to saturated, straight-chain orbranched hydrocarbon radicals containing, in certain embodiments,between one and twenty, including between one and ten, or between oneand six, carbon atoms. Branched means that one or more lower C₁-C₆ alkylgroups such as methyl, ethyl or propyl are attached to a linear alkylchain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl,n-butyl, t-butyl, n-pentyl, and 3-pentyl. Examples of C₁-C₆ alkylradicals include, but are not limited to, methyl, ethyl, propyl,isopropyl, butyl, tert-butyl, neopentyl, n-hexyl radicals; and examplesof C₁-C₈ alkyl radicals include, but are not limited to, methyl, ethyl,propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl,octyl radicals. Examples of C₁-C₂₀ alkyl radicals include but are notlimited to hexadecamethyl, hexadecaethyl, hexadecopropyl,octadecamethyl, octadecaethyl, octadecapropyl and the like.

The term “alkenyl”, as used herein, denotes a monovalent straight orbranched group derived from a hydrocarbon moiety containing, in certainembodiments, from two to six, or two to eight, or two to twenty carbonatoms having at least one carbon-carbon double bond. The double bond mayor may not be the point of attachment to another group. Examples ofC₂-C₈ alkenyl groups include, but are not limited to, for example,ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl andthe like. As defined herein, “akenyl” groups include both cis- andtrans-isomers.

The term “alkynyl”, as used herein, denotes a monovalent straight orbranched group derived from a hydrocarbon moiety containing, in certainembodiments, from two to six, or two to eight, or two to twenty carbonatoms having at least one carbon-carbon triple bond. The triple bond mayor may not be the point of attachment to another group. Examples ofC₂-C₈ alkynyl groups include, but are not limited to, for example,ethynyl, propynyl, butynyl and the like.

The term “alkoxy” refers to an —O-alkyl radical.

The term “thioalkyl” or “alkylthio” refers to an —S-alkyl radical. Insome embodiments, thio group can be replaced by a sulfinyl (SO) orsulfonyl (SO₂).

The terms “hal”, “halo”, or “halogen”, as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The terms “haloalkyl”, “haloalkenyl”, or “haloalkynyl”, as used hereinrefer to an alkyl, alkenyl or alkynyl that is substituted with one ormore halogens or halo groups. Examples of haloalkyl include but are notlimited to CF₃, CH₂CF₃, CCl₃.

The term “aryl”, as used herein, refers to a mono- or poly-cycliccarbocyclic ring system having one or more aromatic rings, fused ornon-fused, including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like. The term aryl includesindoline.

The term “cycloalkyl”, as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic saturated carbocyclic ringcompound. Examples of C₃-C₈-cycloalkyl (3- to 8-membered cycloalkyl)include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclopentyl and cyclooctyl; and examples ofC₃-C₁₂-cycloalkyl include, but not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, bicyclo [2.2.1] heptyl, and bicyclo [2.2.2]octyl and the like.

The term “cycloalkenyl”, as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic partially unsatured (i.e.,non-aromatic) carbocyclic ring compound. In other words, it refers to amonovalent group derived from a monocyclic or polycyclic carbocyclicring compound having at least one carbon-carbon double bond. Examples ofsuch groups include, but are not limited to, cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,and the like.

The term “cycloalkynyl,” as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic partially unsaturated (i.e.,non-aromatic) carbocyclic compound having at least one carbon-carbontriple bond. Examples include cyclooctyne.

The term “heteroaryl”, as used herein, refers to a mono- or poly-cyclic(e.g., bi-, or tri-cyclic or more) fused or non-fused, radical or ringsystem having at least one aromatic ring, having from five to ten ringatoms of which at least one ring atom is selected from S, O, P, and N.In other words, heteroaryl is aryl where containing at least oneheteroatom. Examples of heteroaryl include but are not limited topyridinyl, furanyl, thiazolyl, imidazolyl, indolyl, benzofuranyl, andthe like.

The term “5- or 6-membered heteroaryl”, is taken to mean a ring havingfive to twelve ring atoms of which at least one ring atom is selectedfrom S, O, P, and N. Heteroaryl includes, but is not limited to,pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl,thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl,furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl,quinoxalinyl, and the like.

The term “non-aromatic heterocyclic” ring or “non-aromatic heterocycle,”as used herein, refers to a saturated or unsaturated, non-aromaticmonocyclic or polycyclic, fused or non-fused system, where, for example,at least one ring contains between one and four heteroatomsindependently selected from oxygen, sulfur, phosphorous and nitrogen.The nitrogen and sulfur heteroatoms may optionally be oxidized, and thenitrogen heteroatom may optionally be quaternized. Representativenon-aromatic heterocyclic groups include, but are not limited to,[1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

As used herein, the term “oxo” is understood to describe a carbonylgroup (i.e., C(O)).

As described herein, compounds of the disclosure may optionally besubstituted with one or more substituents, such as those illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the disclosure. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted,”whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituents selected from a specifiedgroup, the substituent may be either the same or different at everyposition.

The term “protected” as described herein, refers to functional groups orcompounds of the present disclosure having a protecting group used insynthesis to temporarily mask the characteristic chemistry of afunctional group (such as hydroxyl, amino, carboxyl, etc.) because itinterferes with another reaction. After completion of the reaction,these protecting groups are removed by common methods, or protectedcompounds are used as prodrugs or as the compounds of the disclosure.

The term “prodrug” or “pharmaceutically acceptable prodrugs”, as usedherein refers to compounds that are rapidly transformed in vivo to yieldthe parent compound, for example by hydrolysis in blood (T. Higuchi andV. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S.Symposium Series; Edward B. Roche, ed., Bioreversible Carriers in DrugDesign, American Pharmaceutical Association and Pergamon Press, 1987,both of which are incorporated herein by reference).

The term “pharmaceutical” or “pharmaceutically acceptable” when usedherein as an adjective, means substantially non-toxic and substantiallynon-deleterious to the recipient. As used herein, the phrase“pharmaceutically acceptable” refers to those compounds, materials,compositions, carriers, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

By “pharmaceutical formulation” it is further meant that the carrier,solvent, excipient(s) and salt must be compatible with the activeingredient of the formulation (e.g. a compound of the disclosure). It isunderstood by those of ordinary skill in this art that the terms“pharmaceutical formulation” and “pharmaceutical composition” aregenerally interchangeable, and they are so used for the purposes of thisapplication and include preparations suitable for administration tomammals, e.g., humans.

A “pharmaceutical composition” as used herein relates to a formulationcontaining a compound of the present disclosure in a form suitable foradministration to a subject. In one embodiment, the pharmaceuticalcomposition is in bulk or in unit dosage form. The unit dosage form isany of a variety of forms, including, for example, a capsule, an IV bag,a tablet, a single pump on an aerosol inhaler or a vial. The quantity ofactive ingredient (e.g., a formulation of the disclosed compound orsalt, hydrate, solvate or isomer thereof) in a unit dose of compositionis an effective amount and is varied according to the particulartreatment involved. One skilled in the art will appreciate that it issometimes necessary to make routine variations to the dosage dependingon the age and condition of the patient. The dosage will also depend onthe route of administration. As used herein, “pharmaceuticallyacceptable carrier” may include any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds such asby producing any undesirable biological effect or otherwise interactingin a deleterious manner with any other component(s) of thepharmaceutical composition, its use is contemplated to be within thescope of this disclosure. Some examples of materials which can serve aspharmaceutically acceptable carriers include, but are not limited to,sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatine; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil, sesame oil; olive oil; corn oil and soybean oil; glycols; such aspropylene glycol; esters such as ethyl oleate and ethyl laurate; agar;buffering agents such as magnesium hydroxide and aluminum hydroxide;alginic acid; pyrogen free water; isotonic saline; Ringer's solution;ethyl alcohol, and phosphate buffer solutions, as well as othernon-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. “Pharmaceuticallyacceptable excipient or carrier” also relates to an excipient or carrierthat is useful in preparing a pharmaceutical composition that isgenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes excipient that is acceptable for veterinaryuse as well as human pharmaceutical use. A “pharmaceutically acceptableexcipient” as used in the specification and claims includes both one andmore than one such excipient.

The compounds disclosed herein include the compounds themselves, as wellas their salts, their solvates, and their prodrugs, if applicable. Asalt, for example, can be formed between an anion and a positivelycharged group (e.g., protonated amino) on a compound of this disclosure.Suitable anions include chloride, bromide, iodide, sulfate, bisulfate,sulfamate, nitrate, phosphate, citrate, methanesulfonate,trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate,succinate, fumarate, tartrate, tosylate, salicylate, lactate,naphthalenesulfonate, and acetate (e.g., trifluroacetate). The term“pharmaceutically acceptable anion” refers to an anion suitable forforming a pharmaceutically acceptable salt. Likewise, a salt can also beformed between a cation and a negatively charged group (e.g.,carboxylate) on a compound of this disclosure. Suitable cations includesodium ion, potassium ion, magnesium ion, calcium ion, and an ammoniumcation such as tetramethylammonium ion. The compounds of this disclosurealso include those salts containing quaternary nitrogen atoms. Examplesof prodrugs include esters and other pharmaceutically acceptablederivatives, which, upon administration to a subject, are capable ofproviding active compounds of this disclosure.

Additionally, physiologically acceptable, i.e. pharmaceuticallycompatible, salts can be salts of the compounds disclosed herein withinorganic or organic acids. Preference is given to salts with inorganicacids, such as, for example, hydrochloric acid, hydrobromic acid,phosphoric acid or sulphuric acid, or to salts with organic carboxylicor sulphonic acids, such as, for example, acetic acid, trifluoroaceticacid, propionic acid, maleic acid, fumaric acid, malic acid, citricacid, tartaric acid, lactic acid, benzoic acid, or methanesulphonicacid, ethanesulphonic acid, benzenesulphonic acid, toluenesulphonic acidor naphthalenedisulphonic acid.

Other pharmaceutically compatible salts which may be mentioned are saltswith customary bases, such as, for example, alkali metal salts (forexample sodium or potassium salts), alkaline earth metal salts (forexample calcium or magnesium salts) or ammonium salts, derived fromammonia or organic amines, such as, for example, diethylamine,triethylamine, ethyldiisopropylamine, procaine, dibenzylamine,N-methylmorpholine, dihydroabietylamine or methylpiperidine.

As used herein, “pharmaceutically acceptable salts” can refer toderivatives of the compounds of the present disclosure wherein theparent compound is modified by making acid or base salts thereof.Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines, alkali or organic salts of acidic residues such as carboxylicacids, and the like. The pharmaceutically acceptable salts include theconventional non-toxic salts or the quaternary ammonium salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. For example, such conventional non-toxic salts include, but arenot limited to, those derived from inorganic and organic acids selectedfrom 2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic,benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts can include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present disclosure alsoencompasses salts formed when an acidic proton present in the parentcompound either is replaced by a metal ion, e.g., an alkali metal ion,or an alkaline earth metal ion, e.g., an aluminum ion; or coordinateswith an organic base such as ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methylglucamine, diethylamine,diethylaminoethanol, ethylenediamine, imidazole, lysine, arginine,morpholine, 2-hydroxyethylmorpholine, dibenzylethylenediamine,trimethylamine, piperidine, pyrrolidine, benzylamine,tetramethylammonium hydroxide and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The compounds of the present disclosure can also be prepared asprodrugs. In certain embodiments, one or more compounds of the presentdisclosure are formulated as a prodrug. In certain embodiments, upon invivo administration, a prodrug is chemically converted to thebiologically, pharmaceutically or therapeutically more active form. Incertain embodiments, prodrugs are useful because they are easier toadminister than the corresponding active form. For example, in certaininstances, a prodrug may be more bioavailable (e.g., through oraladministration) than is the corresponding active form. In certaininstances, a prodrug may have improved solubility compared to thecorresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, a prodrug is produced by modifying apharmaceutically active compound such that the active compound will beregenerated upon in vivo administration. The prodrug can be designed toalter the metabolic stability or the transport characteristics of adrug, to mask side effects or toxicity, to improve the flavor of a drugor to alter other characteristics or properties of a drug. By virtue ofknowledge of pharmacodynamic processes and drug metabolism in vivo,those of skill in this art, once a pharmaceutically active compound isknown, can design prodrugs of the compound (see, e.g., Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392).

Additionally, the compounds of the present disclosure, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

Some of the compounds of the present disclosure may exist in unsolvatedas well as solvated forms such as, for example, hydrates.

“Solvate” means a solvent addition form that contains either astoichiometric or non-stoichiometric amounts of solvent. Some compoundscan have a tendency to trap a fixed molar ratio of solvent molecules inthe crystalline solid state, thus forming a solvate. If the solvent iswater the solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate. In the hydrates, the water moleculesare attached through secondary valencies by intermolecular forces, inparticular hydrogen bridges. Solid hydrates contain water as so-calledcrystal water in stoichiometric ratios, where the water molecules do nothave to be equivalent with respect to their binding state. Examples ofhydrates are sesquihydrates, monohydrates, dihydrates or trihydrates.Equally suitable are the hydrates of salts of the compounds of thedisclosure.

The disclosure also includes metabolites of the compounds describedherein. Metabolites from chemical compounds, whether inherent orpharmaceutical, are formed as part of the natural biochemical process ofdegrading and eliminating the compounds. The rate of degradation of acompound is an important determinant of the duration and intensity ofits action. Profiling metabolites of pharmaceutical compounds, drugmetabolism, is an important part of drug discovery, leading to anunderstanding of any undesirable side effects.

As used herein, the term “treat,” “treating,” or “treatment” meansdecreasing the symptoms, markers, and/or any negative effects of acondition in any appreciable degree in a patient who currently has thecondition. In some embodiments, treatment may be administered to asubject who exhibits only early signs of the condition for the purposeof decreasing the risk of developing the disease, disorder, and/orcondition.

As used herein, the term “prevent,” “prevention,” or “preventing” refersto any method to partially or completely prevent or delay the onset ofone or more symptoms or features of a disease, disorder, and/orcondition. Prevention treatment may be administered to a subject whodoes not exhibit signs of a disease, disorder, and/or condition.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. As used herein, “therapeutically effective amount” canalso mean that amount necessary to make a clinically observedimprovement in the patient. In some embodiments, the composition isformulated such that it comprises an amount that would not cause one ormore unwanted side effects. An effective amount of a pharmaceuticalagent can also mean that which provides an objectively identifiableimprovement as noted by the clinician or other qualified observer. Theprecise effective amount for a subject will depend upon the subject'sbody weight, size, and health; the nature and extent of the condition;and the therapeutic or combination of therapeutics selected foradministration. Therapeutically effective amounts for a given situationcan be determined by routine experimentation that is within the skilland judgment of the clinician.

As used herein, “subject” means a human or animal (in the case of ananimal, more typically a mammal). In one aspect, the subject is a human.In one aspect, the subject is a male. In one aspect, the subject is afemale.

The compounds of the present disclosure can also be prepared as esters,for example, pharmaceutically acceptable esters. For example, acarboxylic acid function group in a compound can be converted to itscorresponding ester, e.g., a methyl, ethyl or other ester. Also, analcohol or hydroxyl group in a compound can be converted to itscorresponding ester, e.g., acetate, propionate, or other esters.

The present disclosure includes new compounds generally represented byFormula I, Formula IA, Formula IB, or Formula II, or pharmaceuticallyacceptable salts thereof, and methods for preparation and uses thereof.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents.

Compounds

In one aspect, the present disclosure relates to compounds of Formula I:

and pharmaceutically acceptable enantiomers, diastereomers, racemates,mixtures, solvates or salts thereof, wherein R_(c) and A are as definedabove.

In one or more embodiments of Formula I, Formula IA or Formula IB, A isselected from A1 through A14, wherein R can be R^(I)R^(IA), or R^(IB):

In one or more embodiments of Formula I, Formula IA or Formula IB, A isselected from A1 through A14, wherein R can be R^(I)R^(IA), or R^(IB):

In one or more embodiments of Formula I, Formula IA or Formula IB, A isselected from A1 through A14, wherein R can be R^(I)R^(IA), or R^(IB):

In one or more embodiments of Formula I, Formula IA or Formula IB, A is

wherein R can be R^(I) or R^(IA).

In one or more embodiments of Formula I, Formula IA or Formula IB, A is

wherein R can be R^(I) or R^(IA).

In one or more embodiments, compounds of Formula I can have one or moreof the following features. R₄ is H, substituted or unsubstituted C₁-C₆alkyl, NH₂, OH, or SH. R₄ can be C₁-C₆ alkyl optionally substituted withone or more halogens. R₄ can be methyl, CH₂X, CHX₂ or CX₃, wherein X ishalogen. R₄ can be pentyl. R₁ can be hydrogen. R₂ and R₃ can eachindependently be hydrogen, CH₃, CH₂X, CHX₂, CX₃, N₃, OH, or NH₂, whereinX is halogen. R₅ can be H, M, C₁-C₆ alkyl, phenyl, or benzyl. M⁺ can beNa⁺, Li⁺, K⁺, Ca²⁺, Mg²⁺, or NR_(g)R_(d)R_(e)R_(f) ⁺, wherein R_(g),R_(d), R_(e) and R_(f) are each independently hydrogen or C₁₋₅ alkyl.R^(I) can be H. When X₂ is absent, v can be 0. R₁ can be hydrogen. R₄₅can be hydrogen. R_(c) can be C₁-C₆ alkyl, e.g., methyl.

In one or more embodiments, of the compounds of Formula I, or FormulaIA, Ri is —H. In one or more embodiments, R₂ is —OH. In one or moreembodiments, R₄ is —OH. In one or more embodiments, R₂ and R₄ are each—OH. In one or more embodiments, R₃ is —H. In one or more embodiments,R₄₄ is —H. In one or more embodiments, R₃ and R₄₄ are each —H. In one ormore embodiments, R^(IA) is —H. In one or more embodiments R_(c) is—CH₃. In one or more embodiments, v is 1, X₂ is —O—, n is 0, and R^(I)is —H. In one or more embodiments, v is 1, X₂ is —O—, n is 0, and R^(IA)is —H.

In one or more embodiments of the compounds of Formula II, R^(a) andR^(b) are both —H. In one or more embodiments, R_(c) is —H. In one ormore embodiments, n is 0. In one or more embodiments, R^(II) is —H. Inone or more embodiments, R^(a), R^(b), and R^(c) are —H. In one or moreembodiments, R^(a), R^(b), and R^(c) are —H and n is 0. In one or moreembodiments, R^(a), R^(b), and R^(c) are —H. In one or more embodiments,R^(a), R^(b), and R^(c) are —H, n is 0 and R^(II) is —H.

In one or more embodiments, the present disclosure provides a compoundof Formula IB:

and pharmaceutically acceptable salts, solvates, enantiomers,diastereomers, racemates or mixtures thereof, wherein:

A is:

X₁ is —CR₁₁R₁₂— or —OCH₂CH₂— wherein the oxygen atom is distal to theR^(IB) moiety in A;

R₁₁ and R₁₂ are independently hydrogen or C₁-C₄ alkyl, wherein the alkylis optionally substituted with one or more halogen, —OH, —SH, or —NH₂;

X₂ is absent, —O—, —C(O)O—, or —OCH₂— wherein the oxygen atom is distalto the R^(IB) moiety in A;

each R^(IB) independently is hydrogen, —C₁-C₆ alkyl,

or R^(IB) is an amino acid residue bound via the carbonyl group, whereinthe alkyl is optionally substituted with one or more halogen, —OH, —SH,or —NH₂;

v is 0 or 1;

n is 0, 1, 2, or 3 and when X₂ is —C(O)O—, n is 0;

p is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11;

-   -   q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        or 18;

R_(z) is hydrogen, halogen, —C₁-C₄ alkylthio, —C₁-C₄ alkoxy, —C₁-C₄alkyl, —C₂-C₄ alkenyl, —C₂-C₄alkynyl, aryl, heteroaryl, —C₃-C₈cycloalkyl, —C₄-C₈ cycloalkenyl, or 3- to 5-membered nonaromaticheterocycle, wherein each alkylthio, alkoxy, alkyl, alkenyl, alkynyl,aryl, heteroaryl, cycloalkyl, cycloalkenyl, or heterocycle is optionallysubstituted with one or more halogen, —OH, —SH, or —NH₂;

R_(a), R_(b), R_(x), and R_(y) are each independently selected from thegroup consisting of hydrogen, halogen, —OH, —SH, —C₁-C₆ alkoxy, aryloxy,—C₁-C₆ alkylthio, arylthio, —OC(O)C₁-C₆ alkyl, —OC(O)aryl, —C₁-C₆ alkyl,—C₂-C₆ alkenyl, —C₂-C₆ alkynyl, aryl, heteroaryl, —C₃-C₈ cycloalkyl, and—C₄-C₈ cycloaklenyl, wherein each alkoxy, aryloxy, alkylthio, arylthio,alkyl, aryl, alkenyl, alkynyl, heteroaryl, cycloalkyl, or cycloalkenylis optionally substituted with one or more halogen, —OH, —SH, or —NH₂;

or any two R_(a) or R_(b), together with the atom to which they are bothattached, can combine to form a C₃-C₈ spirocycloalkyl or 3- to8-membered spiroheterocycle;

or any two R_(a) or R_(b), when on adjacent atoms, can combine to form acis- or trans-carbon-carbon double bond or a carbon-carbon triple bond;

or any two R_(a) or R_(b), when on adjacent atoms, can combine to forman aryl, heteroaryl, —C₃-C₁₀cycloalkyl, —C₄-C₁₀cycloalkenyl, or 5- to10-membered ring heterocycle;

or any CR_(a)R_(b) can be replaced by —O—, —S—, —S(O)—, or —SO₂—;

or any two R_(x) or R_(y), together with the atom to which they are bothattached, can combine to form a —C₃-C₈ spirocycloalkyl or 3- to8-membered spiroheterocycle;

or any two R_(x) or R_(y), when on adjacent atoms, can combine to form acis- or trans-carbon-carbon double bond or a carbon-carbon triple bond;

or any two R_(x) or R_(y), when on adjacent atoms, can combine to forman aryl, heteroaryl, —C₃-C₁₀cycloalkyl, —C₄-C₁₀cycloalkenyl, or 5- to10-membered ring heterocycle;

or any CR_(x)R_(y) can be replaced by —O—, —S—, —S(O)—, or —SO₂—;

R₁ and R₄₅ are each independently hydrogen, halogen, —N₃, —OH, —NH₂,—SH, —C₁-C₆ alkyl, —C₃-C₆ cycloalkyl, —C₂-C₆ alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆ alkynyl, cycloalkynyl, —C₁-C₆ alkoxy, or —C₁-C₆alkylthio wherein each alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, alkoxy or alkylthio is indepdendently substitutedwith one or more halogen, —N₃, —OH, —NH₂, or —SH;

R₂, R₃, R₄ and R₄₄ are each independently hydrogen, halogen, —N₃, —OH,—NH₂, —SH, —C₁-C₆ alkyl, —C₁-C₆ alkoxy, or —C₁-C₆ alkylthio, whereineach alkyl, alkoxy, or alkylthio is optionally substituted with one ormore halogen, —N₃, —OH, —NH₂, or —SH;

or R₃ and one of R₄ and R₄₄, together with the atoms to which they areattached can form a carbon-carbon double bond;

R₅ is independently hydrogen, —R^(IB), M⁺, aryl, aralkyl, —C₁-C₆ alkyl,—C₁-C₆ heteroalkyl, cycloalkyl, non-aromatic heterocyclic ring, orheteroaryl, wherein M⁺ is a cation and wherein each aryl, aralkyl,alkyl, heteroalkyl, cycloalkyl, heterocycle, or heteroaryl is optionallysubstituted with one or more halogen, —N₃, —OH, —NH₂, or —SH, andwherein R₅ is not an amino acid; and

R_(c) is —C₁-C₆ alkyl, —C₃-C₆ cycloalkyl, —C₂-C₆ alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆ alkynyl, cycloalkynyl, or aryl, wherein each alkyl,cycloalkyl, alkenyl, cycloalkenyl, or aryl is optionally substitutedwith one or more halogen, —N₃, —OH, —NH₂, or —SH.

In one or more embodiments, R^(I), R^(IA), R^(B), or R^(II) is selectedfrom:

—H,

In one or more embodiments, the compound is of the Formula I-a:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In one or more embodiments, the compound is of the Formula I-b:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In one or more embodiments, the compound is of the Formula I-c:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In one or more embodiments, the compound is of the Formula I-d:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In one or more embodiments, the compound of the disclosure is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In one or more embodiments, the compound of the disclosure is Compound1:

(Compound 1;4-amino-7-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide),or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In one or more embodiments, the compound of the disclosure is Compound1-triphosphate (Compound 1-TP or Compound 1-PPP), or a pharmaceuticallyacceptable salt, solvate, enantiomer, diastereomer, racemate or mixturethereof:

In one or more embodiments, the compound of the disclosure is selectedfrom:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In one or more embodiments, the compound of the disclosure is selectedfrom:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In one or more embodiments, the compound of the disclosure is selectedfrom:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In one or more embodiments, the compound of the disclosure is selectedfrom:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In one or more embodiments, the compound of the disclosure is selectedfrom:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In some embodiments, the present disclosure provides the use of acompound as described herein, or a pharmaceutically acceptable salt,solvate, enantiomer, diastereomer, racemate or mixture thereof, in themanufacture of a medicament for treating a disease.

In some embodiments, the present disclosure provides the use of thecompound

in the manufacture of a medicament for treating a disease. In someembodiments, the disease is a viral infection. In some embodiments, theviral infection is a norovirus.

In some embodiments, the present disclosure provides the use of acompound as described herein in the treatment of a disease.

In some embodiments, the present disclosure provides the use of thecompound

for the treatment of a disease. In some embodiments, the disease is aviral infection. In some embodiments, the viral infection is norovirusinfection.

In some embodiments, the compound of the disclosure is selected fromcompound 1, 2, 3, 4, 5, 6, 71, 77, 76, 107, 111, 126, 133, 137, 139,141, 143, or 145, or a pharmaceutically acceptable salt, solvate,enantiomer, diastereomer, racemate or mixture thereof, or anycombination thereof.

Methods of Synthesis

The compounds of the present disclosure may be made by a variety ofmethods, including standard chemistry. Suitable synthetic routes aredepicted in the schemes given below.

The compounds described herein (e.g., compounds of Formula I, FormulaIA, Formula IB, or Formula II) may be prepared by methods known in theart of organic synthesis as set forth in part by the following syntheticschemes and examples. In the schemes described below, it is wellunderstood that protecting groups for sensitive or reactive groups areemployed where necessary in accordance with general principles orchemistry. Protecting groups are manipulated according to standardmethods of organic synthesis (T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999). These groups are removed at a convenient stage of the compoundsynthesis using methods that are readily apparent to those skilled inthe art. The selection processes, as well as the reaction conditions andorder of their execution, shall be consistent with the preparation ofcompounds of Formula I, Formula IA, Formula IB, or Formula II

Those skilled in the art will recognize if a stereocenter exists in thecompounds of Formula I, Formula IA, Formula IB, or Formula II.Accordingly, the present disclosure includes both possible stereoisomers(unless specified in the synthesis) and includes not only racemiccompounds but the individual enantiomers and/or diastereomers as well.When a compound is desired as a single enantiomer or diastereomer, itmay be obtained by stereospecific synthesis or by resolution of thefinal product or any convenient intermediate. Resolution of the finalproduct, an intermediate, or a starting material may be affected by anysuitable method known in the art. See, for example, “Stereochemistry ofOrganic Compounds” by E. L. Eliel, S. H. Wilen, and L. N. Mander(Wiley-lnterscience, 1994).

The compounds described herein may be made from commercially availablestarting materials or synthesized using known organic, inorganic, and/orenzymatic processes.

As shown above in Scheme 1,4-chloro-2-methyl-7H-pyrrolo[2,3-d]pyrimidine (a; Scheme 1 numbering)can be iodinated in the presence of N-iodosuccinimide (NIS). Theresulting 4-chloro-5-iodo-2-methyl-7H-pyrrolo[2,3-d]pyrimidine (b) canbe treated with a protected furan (c) as shown in Step 2 to givecompound (d). Radical substitution of (d) gives the corresponding cyanoderivative (e) which can undergo deprotection and nucleophilic aromaticsubstitution at the chlorine-bound carbon to give amine-derivative (f).Finally, nitrile hydration of (f) gives4-amino-7-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide(1).

Step 1:

4-amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile,(3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate and DCE were charged in a reactor. Stirring was started andDBU was added. TMSOTf (8.01 kg) was added slowly. The reaction mixturewas diluted with DCM and quenched slowly with water while being cooled.The reaction was extracted with DCM (19.90 kg), and washed with satNaHCO₃. The aqueous phase was further extracted with DCM (19.71 kg) andwashed with brine.

Step 2:

To a reactor were charged(2R,3R,4R,5R)-2-(4-amino-6-bromo-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate, 10% Pd on C and THF. Hydrogen was submitted to the reactorand the mixture was stirred for about 4 hours at room temperature atabout 31 psi.

The reaction mixture was filtered over Celite (7.2 kg) and a polishfilter and the filter residue was washed with THF. The combined filtrateand wash was transferred to a 100-L jacketed reactor with the aid of aTHF wash. The contents of the reactor were vacuum distilled with amaximum batch temperature of 30.0° C. over a period of about 6 hours toa final volume of 27 L. IPA was charged to the reactor. The contents ofthe reactor were vacuum distilled. IPA was charged to the reactor. Thecontents of the reactor were heated to about 60° C., agitated, andcooled slowly to about 5° C. Cold stirring was continued for a period ofabout 9 h with a minimum temperature of about 1° C. The slurry wasfiltered and washed with IPA. The residue was dried under vacuum with anitrogen bleed to provide an LOD of 0.36%. Yield: (73.9%). ¹H NMRconfirms structure. Purity: 97.78% (HPLC, AUC).

Step 3:

A solution of(2R,3R,4R,5R)-2-(4-amino-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate and THF was heated and the addition of NaOH was started. Theinitial addition gave a biphasic mixture and endothermic response but asthe addition continued a single phased, clear solution formed which wasaccompanied by a fast exotherm; the reaction temperature was maintainedduring the rest of the addition and for an additional ˜2½ h. IPC showedthat no(2R,3R,4R,5R)-2-(4-amino-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate was left.

The reaction mixture was cooled to 21° C. and neutralized with 3 N HClwith external cooling to neutral pH. The mixture continued to cool andthe resulting neutralized mixture was distilled under vacuum until theemergence of solids were observed in the pot. The suspension was cooledand stirred for about 2 h at about 2° C. The beige suspension wasfiltered to afford a dark filtrate; the off-white residue was washedonce with cold water.

In certain embodiments, the compounds of the disclosure, e.g., FormulaI, Formula IA, Formula IB, or Formula II may be prepared as enantiomers,diastereomers, and racemates. In some embodiments, compounds of thedisclosure, e.g., Formula I, Formula IA, Formula IB, or Formula IIinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the disclosure. Unless otherwise stated, alltautomeric forms of the compounds disclosed herein are within the scopeof the disclosure.

Compounds of the disclosure, e.g., Formula I, Formula IA, Formula IB, orFormula II can be synthesized substantially free of impurities.Compounds of the disclosure are more than or equal to about 99% w/wpure. In certain embodiments the phosphonate esters may be prepared on alarge scale, for example on an industrial production scale rather thanon an experimental/laboratory scale. For example, a batch-type processaccording to the methods of the disclosure allows the preparation ofbatches of at least 1 g, or at least 5 g, or at least 10 g, or at least100 g, or at least 1 kg, or at least 100 kg of phosphonate esterproduct. Furthermore, the methods allow the preparation of a phosphonateester product having a purity of at least 98%, or at least 98.5% asmeasured by HPLC. In preferred embodiments, these products are obtainedin a reaction sequence that does not involve purification by any form ofchromatography (e.g., gas chromatography, HPLC, preparative LC, sizeexclusion chromatography, and the like).

Pharmaceutical Compositions and Methods of Treatment

As set forth above, provided herein are pharmaceutical compositionscomprising compounds of the disclosure (e.g., Formula I, Formula IA,Formula IB, or Formula II) or pharmaceutically acceptable salts thereof.In some embodiments, the present disclosure provides pharmaceuticalcompositions comprising compounds of Formula I, Formula IA, Formula IB,or Formula II or pharmaceutically acceptable salts thereof and apharmaceutically acceptable carrier and/or diluent. In some embodimentsthe present disclosure provides compounds of Formula I, Formula IA,Formula IB, or Formula II formulated as a pharmaceutical composition. Inone embodiment, compounds of Formula I, Formula IA, Formula IB, orFormula II is formulated as a tablet. In another embodiment, compoundsof Formula I, Formula IA, Formula IB, or Formula II is formulated as asuspension.

Techniques for formulation and administration of the disclosed compoundscan be found in Remington: the Science and Practice of Pharmacy, 22^(nd)edition, Pharmaceutical Press (2012).

In an embodiment, the compounds described herein, and thepharmaceutically acceptable salts thereof, are used in pharmaceuticalpreparations in combination with a pharmaceutically acceptable carrieror diluent. Suitable pharmaceutically acceptable carriers include inertsolid fillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

The compounds of the disclosure, e.g., compounds of Formula I, FormulaIA, Formula IB, or Formula II or pharmaceutically acceptable saltsthereof described herein may be combined with a pharmaceuticallyacceptable carrier according to conventional pharmaceutical compoundingtechniques. Furthermore, the carrier may take a wide variety of formsdepending on the form of the preparation desired for administration,e.g. oral, nasal, rectal, vaginal, parenteral (including intravenousinjections or infusions). In preparing compositions for oral dosage formany of the usual pharmaceutical media may be employed. Usualpharmaceutical media include, for example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents, and the likein the case of oral liquid preparations (such as for example,suspensions, solutions, emulsions and elixirs); aerosols; or carrierssuch as starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike, in the case of oral solid preparations (such as for example,powders, capsules, and tablets).

In another embodiment, the disclosure provides a method for thetherapeutic and/or prophylactic treatment of viral infection in asubject, e.g., an immunodeficient subject, the method comprisingadministering any one of the compounds of Formula I, Formula IA, FormulaIB, or Formula II or pharmaceutically acceptable salt thereof. In someembodiments, the salt has a purity of equal to or greater than 91% w/w,e.g., having less than or equal to 9% w/w of impurities, to the subject.

Pharmaceutical compositions comprising the compounds of the presentdisclosure (e.g., compounds of Formula I, Formula IA, Formula IB, orFormula II) may be formulated to have any concentration desired. In someembodiments, the composition is formulated such that it comprises atleast a therapeutically effective amount.

Pharmaceutical compositions include those suitable for oral, sublingual,nasal, rectal, vaginal, topical, buccal and parenteral (includingsubcutaneous, intramuscular, and intravenous) administration, althoughthe most suitable route will depend on the nature and severity of thecondition being treated. The compositions may be conveniently presentedin unit dosage form, and prepared by any of the methods well known inthe art of pharmacy. In certain embodiments, the pharmaceuticalcomposition is formulated for oral administration in the form of a pill,capsule, lozenge or tablet. In other embodiments, the pharmaceuticalcomposition is in the form of a suspension.

When the compounds of the present disclosure are administered aspharmaceuticals to mammals, e.g., humans, they can be given per se or asa pharmaceutical composition containing, for example, about 0.1% to99.9%, about 0.2 to 98%, about 0.3% to 97%, about 0.4% to 96%, or about0.5 to 95% of active ingredient in combination with a pharmaceuticallyacceptable carrier. In one embodiment pharmaceutical compositioncontaining about 0.5% to 90% of active ingredient in combination with apharmaceutically acceptable carrier is suitable for administration tomammals, e.g., humans. Some embodiments of the present disclosureprovide preparation of a pharmaceutical composition comprising about0.1% to 99.9%, about 0.2 to 98%, about 0.3% to 97%, about 0.4% to 96%,or about 0.5 to 95% of the compounds of Formula I, Formula IA, FormulaIB, or Formula II or pharmaceutically acceptable salts thereof, e.g.,any one of the Compounds in Table 7 or pharmaceutically acceptable saltthereof, for use in treating, preventing, or prophylaxis of viralinfections or viral infection associated disorders. The presentdisclosure provides use of about 0.1% to 99.9%, about 0.2 to 98%, about0.3% to 97%, about 0.4% to 96%, or about 0.5 to 95% of the compounds ofFormula I, Formula IA, Formula IB, or Formula II or pharmaceuticallyacceptable salts thereof for the manufacture of a medicament containingeffective amounts of the compound for use in treating, preventing, orprophylaxis of viral infections and viral infection associated diseases.

The present disclosure provides for a compound of Formula I, Formula IA,Formula IB, or Formula II for use in treating a viral infection or aviral infection-associated disease or disorder. The compounds can be ina pharmaceutical formulation comprising about 0.1% to 99.9%, about 0.2to 98%, about 0.3% to 97%, about 0.4% to 96%, or about 0.5 to 95% of thecompounds of Formula I, Formula IA, Formula IB, or Formula II.

For any compound, the therapeutically effective amount of a compound orcomposition can be estimated initially either in cell culture assays,e.g., of neoplastic cells, or in animal models, usually rats, mice,rabbits, dogs, or pigs. The animal model may also be used to determinethe appropriate concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. Therapeutic/prophylactic efficacy and toxicitymay be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., ED₅₀ (the dose therapeutically effectivein 50% of the population) and LD₅₀ (the dose lethal to 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The dosage may vary within this range depending upon thedosage form employed, sensitivity of the patient, and the route ofadministration.

The pharmaceutical compositions containing compounds of Formula I,Formula IA, Formula IB, or Formula II of the present disclosure may bemanufactured in a manner that is generally known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.Pharmaceutical compositions may be formulated in a conventional mannerusing one or more pharmaceutically acceptable carriers comprisingexcipients and/or auxiliaries that facilitate processing of the activecompounds into preparations that can be used pharmaceutically. Theappropriate formulation is dependent upon the route of administrationchosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as, for example, peppermint, methyl salicylate, or orangeflavoring.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. In some embodiments, the materials can also beobtained commercially, e.g., from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The compounds of Formula I, Formula IA, Formula IB, or Formula II orpharmaceutically acceptable salts thereof are formulated as apharmaceutical composition or are used in the manufacture of amedicament for the treatment of a viral infection and/or viral infectionassociated disease and/or disorder. Additionally, the present disclosureprovides a compound of Formula I, Formula IA, Formula IB, or Formula II,or a composition comprising a compound of Formula I, Formula IA, FormulaIB, or Formula II for use in treating a viral infection or a viralinfection-associated disease or disorder. The composition and/or themedicament of the compounds of Formula I, Formula IA, Formula IB, orFormula II or pharmaceutically acceptable salts thereof can beformulated as a tablet or suspension. Tablets of the compounds ofFormula I, Formula IA, Formula IB, or Formula II or pharmaceuticallyacceptable salts thereof are formulated comprising pharmacologicallyacceptable buffers, excipients, carriers, including emulsifiers,enhancers (e.g., absorption enhancers), disintegrants (e.g.,Polyvinylpolypyrrolidone (polyvinyl polypyrrolidone, PVPP, crospovidone,crospolividone or E1202), which is a highly cross-linked modification ofpolyvinylpyrrolidone (PVP)), and/or polymers disclosed in the presentdisclosure and well-known in the art.

In one embodiment, the present disclosure provides tablet formulation ofthe compounds of Formula I, Formula IA, Formula IB, or Formula II orpharmaceutically acceptable salts thereof for use in treatment,prophylactic treatment or prevention viral infection and/or viralassociated disease or disorder. The present disclosure provides tabletformulation of the compounds of Formula I, Formula IA, Formula IB, orFormula II or pharmaceutically acceptable salts thereof for use intreating subjects in need of such treatment including but not limited toimmunodeficient subjects, or pre- or post-organ and/or tissuetransplantation subjects. The present disclosure provides the compoundsof Formula I, Formula IA, Formula IB, or Formula II or pharmaceuticallyacceptable salts thereof for the use in the manufacture of a medicamentfor use in treating subjects in need of such treatment including but notlimited to immunodeficient subjects, or pre- or post-organ and/or tissuetransplantation subjects.

In one embodiment, the present disclosure provides suspensionformulations of the compounds of Formula I, Formula IA, Formula IB, orFormula II or pharmaceutically acceptable salts thereof for use inprophylactic treatment or prevention viral infection and/or viralassociated disease and/or disorder. The present disclosure providessuspension formulation of the compounds of Formula I, Formula IA,Formula IB, or Formula II or pharmaceutically acceptable salts thereoffor use in treating subjects in need of such treatment including but notlimited to immunodeficient subjects, or pre- or post-organ and/or tissuetransplantation subjects.

In another embodiment, additional excipients include but are not limitedto sodium phosphate, dibasic, citric acid (monohydrate) (about 0.01-5%wt), sodium citrate (about 0.01-5% wt), xanthum gum (about 0.01-5% wt),methylparaben (sodium salt) (about 0.01-5% wt), propylparaben (sodiumsalt) (about 0.01-5% wt), sucralose (about 0.01-5% wt), microcrystallinecellulose and carboxymethylcellulose sodium (VivaPur MCG 591) (about0.5-10% wt), high fructose corn syrup (about 10-70% wt), lemon limeflavor (WONF220J15) (about 0.01-5% wt), sodium hydroxide pellets, sodiumhydroxide/hydrochloric acid, and purified water (about 68.93% wt).

The formulations of the present disclosure are used in manufacturing amedicament in prophylactic treatment and/or prevention viral infectionand/or viral associated disease and/or disorder.

In another embodiment, the present disclosure provides compositions(e.g., pharmaceutical compositions) with desirable pharmacokineticcharacteristics. For example, the compositions of the disclosure mayprovide a blood level of the compounds of Formula I, Formula IA, FormulaIB, or Formula II or pharmaceutically acceptable salts thereof which,after metabolism to the therapeutically-active form (e.g., thediphosphate equivalent), results in blood levels of the metabolite thatdo not induce toxicity.

In some embodiments, the present disclosure provides a pharmaceuticalcomposition comprising a compound described herein (e.g., compound 1, 2,3, 4, 5, 6, 71, 77, 76, 107, 111, 126, 133, 137, 139, 141, 143, or 145,or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof, or any combination thereof).

Disease Indications

In some embodiments, the present disclosure provides a method oftreating a viral infection or a viral-infection-associated disease ordisorder comprising administering to a subject in need thereof acompound of the disclosure (e.g., compound 1, 2, 3, 4, 5, 6, 71, 77, 76,107, 111, 126, 133, 137, 139, 141, 143, or 145, or a pharmaceuticallyacceptable salt, solvate, enantiomer, diastereomer, racemate or mixturethereof, or any combination thereof).

In some embodiments, the present disclosure provides a use of a compoundof the disclosure (e.g., compound 1, 2, 3, 4, 5, 6, 71, 77, 76, 107,111, 126, 133, 137, 139, 141, 143, or 145, or a pharmaceuticallyacceptable salt, solvate, enantiomer, diastereomer, racemate or mixturethereof, or any combination thereof) in the manufacture of a medicamentfor treating a disease, (e.g., a viral infection or aviral-infection-associated disease or disorder).

In some embodiments, the present disclosure provides a use of a compoundof the disclosure (e.g., compound 1, 2, 3, 4, 5, 6, 71, 77, 76, 107,111, 126, 133, 137, 139, 141, 143, or 145, or a pharmaceuticallyacceptable salt, solvate, enantiomer, diastereomer, racemate or mixturethereof, or any combination thereof) for treating a disease, (e.g., aviral infection or a viral-infection-associated disease or disorder).

The present disclosure provides treatment and/or prevention of a viralinfection with the compounds disclosed herein and pharmaceuticallyacceptable salts thereof. The compounds represented by Formula I,Formula IA, Formula IB, or Formula II are used in treating, preventing,and/or manufacturing a medicament for treating and/or preventing atleast one virus selected from but not limited to ssRNA viruses. In someembodiments, the virus can be a norovirus, human cytomegalovirus (HCMV),BK virus (BKV), Epstein-Barr virus (EBV), adenovirus, JC virus (JCV),SV40, MC virus (MCV), KI virus (KIV), WU virus (WUV), vaccinia, herpessimplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human herpesvirus 6 (HHV-6), human herpes virus 8 (HHV-8), hepatitis B virus,hepatitis C virus, varicella zoster virus (VZV), variola major, variolaminor, smallpox, cowpox, camelpox, monkeypox, poliovirus, picornaviridae(e.g., rhinovirus), paramyxoviridae (e.g., respiratory syncytial virus,RSV), ebola virus, Marburg virus, Epstein-Barr virus (EBV), influenza,enterovirus (e.g., EV68 and EV71, papilloma virus, West Nile virus,yellow fever virus, foot-and-mouth disease virus, Rift Valley fevervirus, and other flavivirus, arenavirus, bunyavirus, alphavirus, andhuman immunodeficiency virus (HIV) infections, and any combinationthereof.

The present disclosure further provides a method of treatment,prevention, or delaying on-set of viral infections orviral-infection-associated diseases or disorders (e.g., norovirus, humancytomegalovirus (HCMV), BK virus (BKV), Epstein-Barr virus (EBV),adenovirus, JC virus (JCV), SV40, MC virus (MCV), KI virus (KIV), WUvirus (WUV), vaccinia, herpes simplex virus 1 (HSV-1), herpes simplexvirus 2 (HSV-2), human herpes virus 6 (HHV-6), human herpes virus 8(HHV-8), hepatitis B virus, hepatitis C virus, varicella zoster virus(VZV), variola major, variola minor, smallpox, cowpox, camelpox,monkeypox, poliovirus, picornaviridae (e.g., rhinovirus),paramyxoviridae (e.g., respiratory syncytial virus, RSV), ebola virus,Marburg virus, Epstein-Barr virus (EBV), influenza, enterovirus (e.g.,EV68 and EV71, papilloma virus, West Nile virus, yellow fever virus,foot-and-mouth disease virus, Rift Valley fever virus, & otherflavivirus, arenavirus, bunyavirus, alphavirus, and humanimmunodeficiency virus (HIV) infections, and any combination thereof) byorally administering to a subject a pharmaceutical compositioncomprising a therapeutically effective dose of a compound of Formula I,Formula IA, Formula IB, or Formula II or a pharmaceutically acceptablesalt thereof, in combination with one or more of compound or compositionselected from an immunosuppressant and/or an antiviral agent.

In some embodiments, the present disclosure also provides a compound ofFormula I, Formula IA, Formula IB, or Formula II in the manufacture of amedicament for the treatment, prevention, or delaying on-set of viralinfections or viral-infection-associated diseases or disorders (e.g.,norovirus, human cytomegalovirus (HCMV), BK virus (BKV), Epstein-Barrvirus (EBV), adenovirus, JC virus (JCV), SV40, MC virus (MCV), KI virus(KIV), WU virus (WUV), vaccinia, herpes simplex virus 1 (HSV-1), herpessimplex virus 2 (HSV-2), human herpes virus 6 (HHV-6), human herpesvirus 8 (HHV-8), hepatitis B virus, hepatitis C virus, varicella zostervirus (VZV), variola major, variola minor, smallpox, cowpox, camelpox,monkeypox, poliovirus, picornaviridae (e.g., rhinovirus),paramyxoviridae (e.g., respiratory syncytial virus, RSV), ebola virus,Marburg virus, Epstein-Barr virus (EBV), influenza, enterovirus (e.g.,EV68 and EV71, papilloma virus, West Nile virus, yellow fever virus,foot-and-mouth disease virus, Rift Valley fever virus, & otherflavivirus, arenavirus, bunyavirus, alphavirus, and humanimmunodeficiency virus (HIV) infections, and any combination thereof) byorally administering to a subject a pharmaceutical compositioncomprising a therapeutically effective dose of a compound of Formula I,Formula IA, Formula IB, or Formula II or a pharmaceutically acceptablesalt thereof, in combination with one or more of compound or compositionselected from an immunosuppressant and/or an antiviral agent.

In some embodiments, the present disclosure also provides a compound ofFormula I, Formula IA, Formula IB, or Formula II for use in treatment,prevention, or delaying on-set of viral infections orviral-infection-associated diseases or disorders (e.g., norovirus, humancytomegalovirus (HCMV), BK virus (BKV), Epstein-Barr virus (EBV),adenovirus, JC virus (JCV), SV40, MC virus (MCV), KI virus (KIV), WUvirus (WUV), vaccinia, herpes simplex virus 1 (HSV-1), herpes simplexvirus 2 (HSV-2), human herpes virus 6 (HHV-6), human herpes virus 8(HHV-8), hepatitis B virus, hepatitis C virus, varicella zoster virus(VZV), variola major, variola minor, smallpox, cowpox, camelpox,monkeypox, poliovirus, picornaviridae (e.g., rhinovirus),paramyxoviridae (e.g., respiratory syncytial virus, RSV), ebola virus,Marburg virus, Epstein-Barr virus (EBV), influenza, enterovirus (e.g.,EV68 and EV71, papilloma virus, West Nile virus, yellow fever virus,foot-and-mouth disease virus, Rift Valley fever virus, & otherflavivirus, arenavirus, bunyavirus, alphavirus, and humanimmunodeficiency virus (HIV) infections, and any combination thereof) byorally administering to a subject a pharmaceutical compositioncomprising a therapeutically effective dose of a compound of Formula I,Formula IA, Formula IB, or Formula II or a pharmaceutically acceptablesalt thereof, in combination with one or more of compound or compositionselected from an immunosuppressant and/or an antiviral agent.

In some embodiments, the present disclosure provides a method oftreatment, prevention, or delaying on-set of Marburg virus infection orMarburg virus infection associated disease or disorder, by oraladministration to a subject in need thereof a pharmaceutical compositionof a therapeutically effective dose of a compound of Formula I, FormulaIA, Formula IB, or Formula II or a pharmaceutically acceptable saltthereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II in the manufacture of amedicament for treatment, prevention, or delaying on-set of Marburgvirus infection or Marburg virus infection associated disease ordisorder, by oral administration to a subject in need thereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II for use in treatment,prevention, or delaying on-set of Marburg virus infection or Marburgvirus infection associated disease or disorder, by oral administrationto a subject in need thereof.

In some embodiments the present disclosure provides a method oftreatment, prevention, or delaying on-set of Ebola virus infection orEbola virus infection associated disease or disorder, by oraladministration to a subject in need thereof a pharmaceutical compositionof a therapeutically effective dose of a compound of Formula I, FormulaIA, Formula IB, or Formula II or a pharmaceutically acceptable saltthereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II in the manufacture of amedicament for treatment, prevention, or delaying on-set of Ebola virusinfection or Ebola virus infection associated disease or disorder, byoral administration to a subject in need thereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II for use in treatment,prevention, or delaying on-set of Ebola virus infection or Ebola virusinfection associated disease or disorder, by oral administration to asubject in need thereof.

In some embodiments the present disclosure provides a method oftreatment, prevention, or delaying on-set of influenza virus infectionor influenza virus infection associated disease or disorder, by oraladministration to a subject in need thereof a pharmaceutical compositionof a therapeutically effective dose of a compound of Formula I, FormulaIA, Formula IB, or Formula II or a pharmaceutically acceptable saltthereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II in the manufacture of amedicament for treatment, prevention, or delaying on-set of influenzavirus infection or influenza virus infection associated disease ordisorder, by oral administration to a subject in need thereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II for use in treatment,prevention, or delaying on-set of influenza virus infection or influenzavirus infection associated disease or disorder, by oral administrationto a subject in need thereof.

In some embodiments the present disclosure provides a method oftreatment, prevention, or delaying on-set of norovirus virus infectionor norovirus virus infection associated disease or disorder, by oraladministration to a subject in need thereof a pharmaceutical compositionof a therapeutically effective dose of a compound of Formula I, FormulaIA, Formula IB, or Formula II or a pharmaceutically acceptable saltthereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II in the manufacture of amedicament for treatment, prevention, or delaying on-set of norovirusvirus infection or norovirus virus infection associated disease ordisorder, by oral administration to a subject in need thereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II for use in treatment,prevention, or delaying on-set of norovirus infection or norovirusinfection associated disease or disorder, by oral administration to asubject in need thereof.

In some embodiments the present disclosure provides a method oftreatment, prevention, or delaying on-set of picornaviridae virusinfection or picornaviridae virus infection associated disease ordisorder, by oral administration to a subject in need thereof apharmaceutical composition of a therapeutically effective dose of acompound of Formula I, Formula IA, Formula IB, or Formula II or apharmaceutically acceptable salt thereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II in the manufacture of amedicament for treatment, prevention, or delaying on-set ofpicornaviridae virus infection or picornaviridae virus infectionassociated disease or disorder, by oral administration to a subject inneed thereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II for use in treatment,prevention, or delaying on-set of picornaviridae virus infection orpicornaviridae virus infection associated disease or disorder, by oraladministration to a subject in need thereof.

In some embodiments the present disclosure provides a method oftreatment, prevention, or delaying on-set of paramyxoviridae virusinfection or paramyxoviridae virus infection associated disease ordisorder, by oral administration to a subject in need thereof apharmaceutical composition of a therapeutically effective dose of acompound of Formula I, Formula IA, Formula IB, or Formula II or apharmaceutically acceptable salt thereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II in the manufacture of amedicament for treatment, prevention, or delaying on-set ofparamyxoviridae virus infection or paramyxoviridae virus infectionassociated disease or disorder, by oral administration to a subject inneed thereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II for use in treatment,prevention, or delaying on-set of paramyxoviridae infection orparamyxoviridae infection associated disease or disorder, by oraladministration to a subject in need thereof.

In some embodiments of the present disclosure provides a method oftreatment, prevention, or delaying on-set of enterovirus infection orenterovirus infection associated disease or disorder, by oraladministration to a subject in need thereof a pharmaceutical compositionof a therapeutically effective dose of a compound of Formula I, FormulaIA, Formula IB, or Formula II or a pharmaceutically acceptable saltthereof.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II in the manufacture of amedicament for treatment, prevention, or delaying on-set of enterovirusinfection or enterovirus infection associated disease or disorder, byoral administration to a subject in need thereof.

In any of the above embodiments, the compound can be compound 1.

In some embodiments, the present disclosure provides a compound ofFormula I, Formula IA, Formula IB, or Formula II for use in treatment,prevention, or delaying on-set of enterovirus infection or enterovirusinfection associated disease or disorder, by oral administration to asubject in need thereof.

The present disclosure further provides a method of prophylactictreatment, prevention, or delaying on-set of norovirus infection or anorovirus infection associated disease or disorder, by orallyadministering to a subject a pharmaceutical composition comprising atherapeutically effective dose of a compound of Formula I, Formula IA,Formula IB, or Formula II (e.g., compound 1) or a pharmaceuticallyacceptable salt thereof, in combination with one or more antiviralagent. In some embodiments, the method of prophylactic treatmentcomprises treating a subject with a compound of the disclosure prior toinfection with the norovirus.

The present disclosure also provides a compound of Formula I, FormulaIA, Formula IB, or Formula II (e.g., compound 1) in the manufacture of amedicament for the prophylactic treatment, prevention, or delayingon-set of norovirus infection or a norovirus infection associateddisease or disorder, by orally administering to a subject apharmaceutical composition comprising a therapeutically effective doseof a compound of Formula I, Formula IA, Formula IB, or Formula II or apharmaceutically acceptable salt thereof, in combination with one ormore antiviral agent.

The present disclosure also provides a compound of Formula I, FormulaIA, Formula IB, or Formula II (e.g., compound 1) for use in theprophylactic treatment, prevention, or delaying on-set of norovirusinfection or a norovirus infection associated disease or disorder, byorally administering to a subject a pharmaceutical compositioncomprising a therapeutically effective dose of a compound of Formula I,Formula IA, Formula IB, or Formula II or a pharmaceutically acceptablesalt thereof, in combination with one or more antiviral agent. Thepresent disclosure further provides a method of prophylactic treatment,prevention, or delaying on-set of enterovirus infection or anenterovirus infection associated disease or disorder, by orallyadministering to a subject a pharmaceutical composition comprising atherapeutically effective dose of a compound of Formula I, Formula IA,Formula IB, or Formula II or a pharmaceutically acceptable salt thereof,in combination with one or more antiviral agent.

The present disclosure also provides the use of a compound of Formula I,Formula IA, Formula IB, or Formula II in the manufacture of a medicamentfor prophylactic treatment, prevention, or delaying on-set ofenterovirus infection or an enterovirus infection associated disease ordisorder, by orally administering to a subject a pharmaceuticalcomposition comprising a therapeutically effective dose of a compound ofFormula I, Formula IA, Formula IB, or Formula II or a pharmaceuticallyacceptable salt thereof, in combination with one or more antiviralagent.

The present disclosure also provides a compound of Formula I, FormulaIA, Formula IB, or Formula II for use in the prophylactic treatment,prevention, or delaying on-set of enterovirus infection or anenterovirus infection associated disease or disorder, by orallyadministering to a subject a pharmaceutical composition comprising atherapeutically effective dose of a compound of Formula I, Formula IA,Formula IB, or Formula II or a pharmaceutically acceptable salt thereof,in combination with one or more antiviral agent.

In one of the embodiments, compounds of Formula I, Formula IA, FormulaIB, or Formula II are be used to treat norovirus. In anotherembodiments, compounds of Formula I, Formula IA, Formula IB, or FormulaII are used to treat norovirus associated with specific genotypes suchas those in genogroups I, II and IV, VI and VII which are known toinfect humans (Phan et al., J. Med. Virol. 2007 September; 79(9):1388-1400).

Dosage Regimens

The regimen of administration can affect what constitutes apharmaceutically effective amount. The compounds of Formula I, FormulaIA, Formula IB, or Formula II or pharmaceutically acceptable saltsthereof can be administered to the subject either prior to or after theonset of a disease. Further, several divided dosages, as well asstaggered dosages can be administered daily or sequentially, or the dosecan be continuously infused, or can be a bolus injection. Further, thedosages can be proportionally increased or decreased as indicated by theexigencies of the therapeutic or prophylactic situation. Further, thedosages may be co-administered in combination with other antiviral.

The dosage regimen utilizing the compounds can also be selected inaccordance with a variety of factors including type, species, age,weight, sex and medical condition of the patient; the severity of thecondition to be treated; the route of administration; the renal andhepatic function of the patient; and the particular compound or saltthereof employed. An ordinarily skilled physician or veterinarian canreadily determine and prescribe the effective amount of the drugrequired to prevent, counter or arrest the progress of the condition.

In some embodiments, the subject treated for a viral infection (e.g., anorovirus infection or a norovirus infection associated disease ordisorder) is administered once or twice a week with about 40 mg, 50 mg,75 mg, 100 mg, 150 mg, 175 mg, 200 mg, or 250 mg of a compound ofFormula I, Formula IA, Formula IB, or Formula II or a pharmaceuticallyacceptable salt thereof. The present disclosure provides treatment of asubject for norovirus infection or norovirus infection associateddisease or disorder by administering to the subject once a week (QW)about 200 mg or twice a week (BIW) about 100 mg of a compound of FormulaI, Formula IA, Formula IB, or Formula II, or a pharmaceuticallyacceptable salt thereof. In one embodiment, the subject is treated twicea week (BIW) with about 100 mg of the compound. In another embodiment,the subject is treated once a week (QW) with about 200 mg, or twice aweek (BIW) with about 100 mg of the compound.

In an embodiment, a compound of Formula I, Formula IA, Formula IB, orFormula II or pharmaceutically acceptable salt thereof having a purityof equal to or greater than about 91% is administered orally to asubject, for example, at a dosage of about 0.01 mg/kg to about 10 mg/kgor more, e.g., up to 100 mg/kg, or up to 400 mg/kg, or up to 1000 mg/kg.

In another embodiment, a compound of Formula I, Formula IA, Formula IB,or Formula II or pharmaceutically acceptable salt thereof having apurity of equal to or greater than about 91% w/w is administered to asubject at a dosage of about 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, or 10 mg/kg or more orany range therein.

In a preferred aspect, the disease or condition to be treated is viralinfection.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, once every two weeks, or monthly depending on half-life andclearance rate of the particular formulation.

In some embodiments, the administration continues for ten total doses.For instance, the compounds of Formula I, Formula IA, Formula IB, orFormula II can be administered at dosages of about 100 mg twice a weekfor five weeks (i.e., ten total doses). Alternatively, the compounds ofFormula I, Formula IA, Formula IB, or Formula II may be administeredwith a loading dose of about 200 mg followed by about 100 mg dosescontinuing twice a week. In some embodiments, the administrationcontinues for ten total doses. For instance, the compounds of Formula I,Formula IA, Formula IB, or Formula II may be administered at a loadingdose of about 200 mg followed by nine additional about 100 mg dosestwice a week for a total of ten doses. In one of the embodimentsCompounds of Formula I, Formula IA, Formula IB, or Formula II can bedosed daily in the range of about 20-200 mg/day or weekly in the rangeof about 200 mg-3000 mg.

In one or more embodiments the compounds of the disclosure are useful attreating a viral infection such as a norovirus infection or anorovirus-infection associated disease or disorder. In some embodiments,treatment of the infection, e.g., norovirus infection, can comprisedaily dosing, or dosing multiple times per day. In some embodiments, thetotal treatment regimen only lasts as long as the norovirus infection isactive (e.g., between 1-3 days). In some embodiments, the compounds ofthe disclosure can be dosed multiple times per day for 1-3 days to treata norovirus infection.

In another embodiment, tablets or suspensions of the compounds ofFormula I, Formula IA, Formula IB, or Formula II or pharmaceuticallyacceptable salts thereof are administered at a dose of about 40-3000 mgdaily, BID, TID, once a week (QW) or twice a week (BIW). In anotherembodiment, tablets or suspensions of the compounds of Formula I,Formula IA, Formula IB, or Formula II or pharmaceutically acceptablesalts thereof are administered at a dose of about 40-400 mg daily, BID,TID, once a week (QW) or twice a week (BIW).

In therapeutic applications, the dosages of the pharmaceuticalcompositions disclosed herein vary depending on the agent, the age,weight, and clinical condition of the recipient patient, and theexperience and judgment of the clinician or practitioner administeringthe therapy, among other factors affecting the selected dosage. Dosagescan range from about 0.01 mg/kg to about 100 mg/kg. In preferredaspects, dosages can range from about 0.1 mg/kg to about 10 mg/kg. In anaspect, the dose will be in the range of about 1 mg to about 1 g; about10 mg to about 500 mg; about 20 mg to about 400 mg; about 40 mg to about400 mg; or about 50 mg to about 400 mg, in single, divided, orcontinuous doses (which dose may be adjusted for the patient's weight inkg, body surface area in m², and age in years). In certain embodiments,the amount per dosage form can be about 0.1 mg to about 3000 mg, e.g.,about 0.1 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg,about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg,about 95 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg,about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg,about 1000 mg, about 1250 mg, 1500 mg, about 1750 mg, about 2000 mg,about 2500 mg, or about 3000 mg. In one embodiment, the amount can beabout 20 mg. In one embodiment, the amount can be about 50 mg. Inanother embodiment the dosage can be 100 mg. In another embodiment thedose can be 500 mg.

In another embodiment, the compounds of Formula I, Formula IA, FormulaIB, or Formula II or pharmaceutically acceptable salts thereof areadministered to a subject as a single dose. In another embodiment, thecompounds of Formula I, Formula IA, Formula IB, or Formula II orpharmaceutically acceptable salts thereof are administered to a subjectin multiple doses. Multiple doses can be administered regularly, forexample, once every 12 hours, once a day, every 2 days, every 3 days,every 4 days, every 5 days, every 6 days, every 7 days, every 8 days,every 9 days, every 10 days, every 11 days, every 12 days, every 13days, every 14 days or every 15 days. For example, doses can beadministered twice per week. Moreover, each individual dose can beadministered with the same or a different dosage.

For example, a subject can be administered any one the compounds ofFormula I, Formula IA, Formula IB, or Formula II or pharmaceuticallyacceptable salts thereof with a first dose of about 1-20 mg/kg (e.g.,about 1-1.1 mg/kg, about 1.1-1.2 mg/kg, about 1.2-1.3 mg/kg, about1.3-1.4 mg/kg, about 1.4-1.5 mg/kg, about 1.5-1.6 mg/kg, about 1.6-1.7mg/kg, about 1.7-1.8 mg/kg, about 1.8-1.9 mg/kg, about 1.9-2.0 mg/kg,about 2.0-2.1 mg/kg, about 2.1-2.2 mg/kg, about 2.2-2.3 mg/kg, about2.3-2.4 mg/kg, about 2.4-2.5 mg/kg, about 2.5-2.6 mg/kg, about 2.6-2.7mg/kg, about 2.7-2.8 mg/kg, about 2.8-2.9 mg/kg, about 2.9-3.0 mg/kg,about 3.0-3.1 mg/kg, about 3.1-3.2 mg/kg, about 3.2-3.3 mg/kg, about3.3-3.4 mg/kg, about 3.4-3.5 mg/kg, about 3.5-3.6 mg/kg, about 3.6-3.7mg/kg, about 3.7-3.8 mg/kg, about 3.8-3.9 mg/kg, about 3.9-4.0 mg/kg,about 4.0-5.0 mg/kg, about 5.0-6.0 mg/kg, about 6.0-7.0 mg/kg, about7.0-8.0 mg/kg, about 8.0-9.0 mg/kg, about 9.0-10.0 mg/kg, or about 10-20mg/kg) of any one of the Compounds of Formula I, Formula IA, Formula IB,or Formula II (or a pharmaceutically acceptable salt thereof) followedby one or more additional doses at 1-4 mg/kg (e.g., about 1-1.1 mg/kg,about 1.1-1.2 mg/kg, about 1.2-1.3 mg/kg, about 1.3-1.4 mg/kg, about1.4-1.5 mg/kg, about 1.5-1.6 mg/kg, about 1.6-1.7 mg/kg, about 1.7-1.8mg/kg, about 1.8-1.9 mg/kg, about 1.9-2.0 mg/kg, about 2.0-2.1 mg/kg,about 2.1-2.2 mg/kg, about 2.2-2.3 mg/kg, about 2.3-2.4 mg/kg, about2.4-2.5 mg/kg, about 2.5-2.6 mg/kg, about 2.6-2.7 mg/kg, about 2.7-2.8mg/kg, about 2.8-2.9 mg/kg, about 2.9-3.0 mg/kg, about 3.0-3.1 mg/kg,about 3.1-3.2 mg/kg, about 3.2-3.3 mg/kg, about 3.3-3.4 mg/kg, about3.4-3.5 mg/kg, about 3.5-3.6 mg/kg, about 3.6-3.7 mg/kg, about 3.7-3.8mg/kg, about 3.8-3.9 mg/kg, or about 3.9-4.0 mg/kg) of any one of theCompounds of Formula I, Formula IA, Formula IB, or Formula II (or apharmaceutically acceptable salt thereof) in the same week or in thefollowing week. For example, a subject can be administered with a firstdose of about 3 mg/kg followed by one or more additional doses at about1 mg/kg. For example, a subject can be administered with a first dose ofabout 2 mg/kg followed by one or more additional doses at about 3 mg/kg.For example, a subject can be administered with a first dose of 4 mg/kgfollowed by one or more additional doses at about 4 mg/kg.

Multiple doses can also be administered at variable time intervals. Forexample, the first 2, 3, 4, 5, 6, 7, or 8 or more doses can beadministered at an interval of 6 days followed by additional dosesadministered at an interval of 7 days. For example, the first 2, 3, 4,5, 6, 7, or 8 or more doses can be administered at an interval of 7 daysfollowed by additional doses administered at an interval of 3 days.

In one embodiment, the compounds of Formula I, Formula IA, Formula IB,or Formula II or pharmaceutically acceptable salts thereof areadministered to a subject once a week at a dose of about 40-3000 mg, ortwice a week at a dose of about 40-3000 mg.

In some embodiments, the pharmaceutical composition of the presentdisclosure is administered daily, BID, TID, once a week (QW), or twice aweek (BIW) with about 40-3000 mg of compounds of Formula I, Formula IA,Formula IB, or Formula II or pharmaceutically acceptable salts thereof.The pharmaceutical compositions of the present disclosure isadministered daily, BID, TID, once a week (QW), or twice a week (BIW)with about 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 175 mg, 200 mg, 250 mg,275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 450 mg, 500 mg, 500-600mg, 600-700 mg, 700-800 mg, 800-900 mg, or 900-1000 mg, or twice a week(BIW) with about 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 175 mg, 200 mg,250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, or 400 mg, 450 mg, 500mg, 500-600 mg, 600-700 mg, 700-800 mg, 800-900 mg, or 900-1000 mg ofCompounds of Formula I, Formula IA, Formula IB, or Formula II orpharmaceutically acceptable salts thereof.

The present disclosure provides compounds of Formula I, Formula IA,Formula IB, or Formula II administered at a dose of about 1-100 mg/kg(e.g., 10-20 mg/kg, 20-50 mg/kg, 50-75 mg/kg, 75-100 mg/kg).

Routes of Administration

The compounds of the present disclosure, or pharmaceutically acceptablesalts, esters or derivatives thereof, can be administered orally,nasally, intranasally, transdermally, pulmonary, inhalationally,buccally, sublingually, intraperintoneally, subcutaneously,intramuscularly, intravenously, rectally, intrapleurally, intrathecallyand parenterally. In one embodiment, the compound is administeredorally. One skilled in the art will recognize the advantages of certainroutes of administration.

Dosage forms for the topical or transdermal administration of a compoundof this disclosure include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. In one embodiment, theactive compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives, buffersor propellants that are required.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Combination Therapy

The present disclosure provides methods of preventing or treating aviral infection in a subject (e.g., a norovirus infection). The methodscomprise administering a subject a therapeutically effective amount of acompound described herein. The compounds may be used in a monotherapy orcombination therapy regime.

As used herein, “monotherapy” means or refers to the administration of asingle active or therapeutic compound (e.g., a compound of Formula I,Formula IA, Formula IB, or Formula II) to a subject in need thereof.Preferably, monotherapy will involve administration of a therapeuticallyeffective amount of an active compound. For example, norovirusmonotherapy with one of the compound of the present disclosure, or apharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, to a subject in need of treatment of norovirus.Monotherapy may be contrasted with combination therapy, in which acombination of multiple active compounds is administered, preferablywith each component of the combination present in a therapeuticallyeffective amount. In one aspect, monotherapy with a compound of thepresent disclosure, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, is more effective thancombination therapy in inducing a desired biological effect.

As used herein, “combination therapy” or “co-therapy” includes theadministration of a compound of the present disclosure, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, and at least a second agent as part of a specifictreatment regimen intended to provide the beneficial effect from theco-action of these therapeutic agents. The beneficial effect of thecombination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days or weeks depending upon the combination selected).“Combination therapy” may be, but generally is not, intended toencompass the administration of two or more of these therapeutic agentsas part of separate monotherapy regimens that incidentally andarbitrarily result in the combinations of the present disclosure.

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, wherein each therapeuticagent is administered at a different time, as well as administration ofthese therapeutic agents, or at least two of the therapeutic agents, ina substantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example, by administering to thesubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents.Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, oral routes, intravenous routes, intramuscularroutes, and direct absorption through mucous membrane tissues. Thetherapeutic agents can be administered by the same route or by differentroutes. For example, a first therapeutic agent of the combinationselected may be administered by intravenous injection while the othertherapeutic agents of the combination may be administered orally.Alternatively, for example, all therapeutic agents may be administeredorally or all therapeutic agents may be administered by intravenousinjection. The sequence in which the therapeutic agents are administeredis not narrowly critical.

“Combination therapy” also embraces the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies. Where thecombination therapy further comprises a non-drug treatment, the non-drugtreatment may be conducted at any suitable time so long as a beneficialeffect from the co-action of the combination of the therapeutic agentsand non-drug treatment is achieved. For example, in appropriate cases,the beneficial effect is still achieved when the non-drug treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

In some embodiments the present disclosure provides a method oftreatment, prevention, or delaying on-set of a viral infection, (e.g.,norovirus virus infection or norovirus virus infection associateddisease or disorder; influenza virus infection or influenza virusinfection associated disease or disorder), by oral administration to asubject in need thereof a pharmaceutical composition of atherapeutically effective dose of a compound of Formula I, Formula IA,Formula IB, or Formula II or a pharmaceutically acceptable salt thereofin combination with one or more antiviral agent.

The present disclosure also provides a compound of Formula I, FormulaIA, Formula IB, or Formula II in the manufacture of a medicament fortreatment, prevention, or delaying on-set of a viral infection, (e.g.,norovirus virus infection or norovirus virus infection associateddisease or disorder; influenza virus infection or influenza virusinfection associated disease or disorder), by oral administration to asubject in need thereof a pharmaceutical composition of atherapeutically effective dose of a compound of Formula I, Formula IA,Formula IB, or Formula II or a pharmaceutically acceptable salt thereofin combination with one or more antiviral agent.

The present disclosure also provides a compound of Formula I, FormulaIA, Formula IB, or Formula II for use in treatment, prevention, ordelaying on-set of a viral infection, (e.g., norovirus virus infectionor norovirus virus infection associated disease or disorder; influenzavirus infection or influenza virus infection associated disease ordisorder), by oral administration to a subject in need thereof apharmaceutical composition of a therapeutically effective dose of acompound of Formula I, Formula IA, Formula IB, or Formula II or apharmaceutically acceptable salt thereof in combination with one or moreantiviral agent.

In some embodiments the present disclosure provides a method oftreatment, prevention, or delaying on-set of picornaviridae virusinfection or picornaviridae virus infection associated disease ordisorder, by oral administration to a subject in need thereof apharmaceutical composition of a therapeutically effective dose of acompound of Formula I, Formula IA, Formula IB, or Formula II or apharmaceutically acceptable salt thereof in combination with one or moreantiviral agent.

The present disclosure also provides a compound of Formula I, FormulaIA, Formula IB, or Formula II in the manufacture of a medicament fortreatment, prevention, or delaying on-set of a picornaviridae virusinfection or picornaviridae virus infection associated disease ordisorder by oral administration to a subject in need thereof apharmaceutical composition of a therapeutically effective dose of acompound of Formula I, Formula IA, Formula IB, or Formula II or apharmaceutically acceptable salt thereof in combination with one or moreantiviral agent.

The present disclosure also provides a compound of Formula I, FormulaIA, Formula IB, or Formula II for use in treatment, prevention, ordelaying on-set of a picornaviridae virus infection or picornaviridaevirus infection associated disease or disorder by oral administration toa subject in need thereof a pharmaceutical composition of atherapeutically effective dose of a compound of Formula I, Formula IA,Formula IB, or Formula II or a pharmaceutically acceptable salt thereofin combination with one or more antiviral agent.

In some embodiments the present disclosure provides a method oftreatment, prevention, or delaying on-set of paramyxoviridae virusinfection or paramyxoviridae virus infection associated disease ordisorder, by oral administration to a subject in need thereof apharmaceutical composition of a therapeutically effective dose of acompound of Formula I, Formula IA, Formula IB, or Formula II or apharmaceutically acceptable salt thereof in combination with one or moreantiviral agent.

The present disclosure also provides a compound of Formula I, FormulaIA, Formula IB, or Formula II in the manufacture of a medicament fortreatment, prevention, or delaying on-set of a paramyxoviridae virusinfection or paramyxoviridae virus infection associated disease ordisorder by oral administration to a subject in need thereof apharmaceutical composition of a therapeutically effective dose of acompound of Formula I, Formula IA, Formula IB, or Formula II or apharmaceutically acceptable salt thereof in combination with one or moreantiviral agent.

The present disclosure also provides a compound of Formula I, FormulaIA, Formula IB, or Formula II for use in treatment, prevention, ordelaying on-set of a paramyxoviridae virus infection or paramyxoviridaevirus infection associated disease or disorder by oral administration toa subject in need thereof a pharmaceutical composition of atherapeutically effective dose of a compound of Formula I, Formula IA,Formula IB, or Formula II or a pharmaceutically acceptable salt thereofin combination with one or more antiviral agent.

In one embodiment, the method of treating a viral infection, e.g.,influenza virus infection or norovirus infection further comprisesadministering at least one additional antiviral agent. In oneembodiment, the compound of Formula I, Formula II, or Formula IA can befor use in combination with an additional antiviral agent. In one ormore embodiments, the compound of Formula I, Formula IA, Formula IB, orFormula II for use in the manufacture of a medicament in combinationwith an additional antiviral agent. In one embodiment, the oneadditional antiviral agent is an adamantane. In a further embodiment,the one additional antiviral agent is amantadine or rimantadine. Inanother embodiment, the one additional antiviral agent is aneuraminidase inhibitor (e.g., oseltamivir, zanamivir, laninamivir, andperamivir). In a further embodiment, the one additional antiviral agentis oseltamivir or zanamivir.

In some embodiments, the pharmaceutical composition of the presentdisclosure (e.g., a compound of Formula I, Formula IA, Formula IB, orFormula II) is administered in combination with one or more compounds orcompositions selected from midazolam, cyclosporine A, tacrolimus,ganciclovir, valganciclovir, foscavir, cidofovir, second-line anti-CMVdrugs, second-line anti-HCV drugs, foscarnet, filgrastim, pegfilgrastim,corticosteroids such as budesonide, beclomethasone, and broad-spectrumCYP inhibitor aminobenzotriazole or combinations thereof.

In additional embodiments, the compound is for administration incombination with at least one other immunosuppressant agent. In oneembodiment, the immunosuppressant agent is concurrently or sequentiallyadministered. The immunosuppressant agents include but are not limitedto Daclizumab, Basiliximab, Tacrolimus, Sirolimus, Mycophenolate,Cyclosporine A, Glucocorticoids, Anti-CD3 monoclonal antibodies,Antithymocyte globulin, Anti-CD52 monoclonal antibodies, Azathioprine,Everolimus, Dactinomycin, Cyclophosphamide, Platinum, Nitrosurea,Methotrexate, Mercaptopurine, Muromonab, IFN gamma, Infliximab,Etanercept, Adalimumab, Natalizumab, Fingolimod, and combinationsthereof.

The compounds or compositions provided herein may also be used incombination with an enhancer agent, with other active ingredients, orwith an immunosuppressant agent. In certain embodiments, the compoundsmay be administered in combination, or sequentially, with anothertherapeutic agent or an enhancer. Such other therapeutic agents includethose known for treatment, prevention, or amelioration of one or moresymptoms associated with viral infections. It should be understood thatany suitable combination of the compounds provided herein with one ormore of the above-mentioned compounds and optionally one or more furtherpharmacologically active substances are considered to be within thescope of the present disclosure. In another embodiment, the compoundprovided herein is administered prior to or subsequent to the one ormore additional active ingredients. In one embodiment, two or more ofthe antiviral agents disclosed herein are administered serially or incombination. The amount of some enhancers can be selected using methodsknown in the art to enhance the bioavailability of the anti-viral agent.Any amount can be used that provides a desired response by someenhancers. The dosages may range, in a non-limiting example, from 0.001mg to about 3000 mg of compound per kilogram of body weight per day,e.g., 0.01 to 500 mg/kg, or e.g., 0.1-20 mg/kg.

The pharmacokinetic behavior of a composition will vary somewhat fromsubject to subject within a population. The numbers described above forthe compositions disclosed herein are based on the average behavior in apopulation. The present disclosure is intended to encompass compositionsthat on average fall within the disclosed ranges, even though it isunderstood that certain subjects may fall outside of the ranges.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. The presentdisclosure provides a kit including, in addition to a pharmaceuticalcomposition of any one of the disclosed compounds, a container, pack, ordispenser together with instructions for administration.

A compound of the present disclosure, or a pharmaceutically acceptablesalt, prodrug, metabolite, analog or derivative thereof, may beadministered in combination with a second antiviral compound. Forexample, as noted above, the compositions of the present disclosure mayinclude the compounds as described above in combination with one or more(e.g., 1, 2, 3) additional active agents such as described in thissection in analogous manner as known in the art. Additional antiviralactive agents that may be used with the compounds of the presentdisclosure in carrying out the present methods include, but are notlimited to, those that target the M2 ion channel in influenza A viruses(e.g., the adamantanes, such as amantadine and rimantadine); those thatinhibit viral uncoating following entry into the cell, agents that blockrelease of the newly formed virions from the surface of infected cells(e.g., the neuraminidase inhibitors, such as oseltamivir and zanamivir).

Methods for Preventing disease or disorder due to Virus Reactivation.

The current disclosure also provides a method of preventing a disease ordisorder in a subject at risk of virus infection reactivation, by orallyadministering to the subject a pharmaceutical composition of atherapeutically effective dose of a compound of Formula I, Formula IA,Formula IB, or Formula II or a pharmaceutically acceptable salt thereof.In some embodiments, the virus at risk of reactivation can be influenza,norovirus, EBV, ebola, picornaviridae, paramyxoviridae, and Marburgvirus. In some preferred embodiments, the virus at risk of reactivationcan be influenza.

Effect of Food

In some embodiments, the pharmaceutical composition of the currentembodiments, e.g., tablet or suspension, may be provided to a subjectwhen the subject is either fasted or in fed conditions. In oneembodiment, the composition comprising a compound of Formula I, FormulaIA, Formula IB, or Formula II (or a pharmaceutically acceptable saltthereof) may be provided to a subject having an empty stomach, e.g.,after fasting for less than 24 hours but more than 12 hours, more than11 hours, more than 10 hours, more than 8 hours, or more than 5 hours.

In other embodiments, the composition comprising a compound of FormulaI, Formula IA, Formula IB, or Formula II (or a pharmaceuticallyacceptable salt thereof) may be provided to a subject in combinationwith food or subsequent to having food. In one embodiment, a compound ofFormula I, Formula IA, Formula IB, or Formula II (or a pharmaceuticallyacceptable salt thereof) may be taken by a subject on an empty stomach.

Patient Population

In certain embodiments, compounds of Formula I, Formula IA, Formula IB,or Formula II (or a pharmaceutically acceptable salt thereof), acomposition comprising a compound of Formula I, Formula IA, Formula IB,or Formula II, or a combination therapy comprising a composition ofFormula I, Formula IA, Formula IB, or Formula II is administered to amammal in need thereof (e.g., a human) which is about 1 to 6 months old,6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 10 to 15 yearsold, 15 to 20 years old, 20 to 25 years old, 25 to 30 years old, 30 to35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50 yearsold, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 yearsold, 85 to 90 years old, 90 to 95 years old, or 95 to 100 years old. Insome embodiments, the mammal is suffering from a viral infection (e.g.,an ssRNA infection such as a norovirus infection).

In certain embodiments, a compound of Formula I, Formula IA, Formula IB,or Formula II, a composition comprising a compound of Formula I, FormulaIA, Formula IB, or Formula II, or a combination therapy comprising acompound of Formula I, Formula IA, Formula IB, or Formula II isadministered to a human at risk fordeveloping a virus infection. Incertain embodiments, a compound of Formula I, Formula IA, Formula IB, orFormula II, a composition comprising a compound of Formula I, FormulaIA, Formula IB, or Formula II, or a combination therapy comprising acompound of Formula I, Formula IA, Formula IB, or Formula II isadministered to a human with a virus infection. In certain embodiments,the patient is a human about 1 to 6 months old, 6 to 12 months old, 1 to5 years old, 5 to 10 years old, 5 to 12 years old, 10 to 15 years old,15 to 20 years old, 13 to 19 years old, 20 to 25 years old, 25 to 30years old, 20 to 65 years old, 30 to 35 years old, 35 to 40 years old,40 to 45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60years old, 60 to 65 years old, 65 to 70 years old, 70 to 75 years old,75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95years old or 95 to 100 years old.

In some embodiments, a compound of Formula I, Formula IA, Formula IB, orFormula II, a composition comprising a compound of Formula I, FormulaIA, Formula IB, or Formula II, or a combination therapy comprising acompound of Formula I, Formula IA, Formula IB, or Formula II isadministered to a human infant. In other embodiments, a compound ofFormula I, Formula IA, Formula IB, or Formula II, or a combinationtherapy comprising a compound of Formula I, Formula IA, Formula IB, orFormula II is administered to a human child. In other embodiments, acompound of Formula I, Formula IA, Formula IB, or Formula II, acomposition comprising a compound of Formula I, Formula IA, Formula IB,or Formula II, or a combination therapy comprising a compound of FormulaI, Formula IA, Formula IB, or Formula II is administered to a humanadult. In yet other embodiments, a compound of Formula I, Formula IA,Formula IB, or Formula II, a composition comprising a compound ofFormula I, Formula IA, Formula IB, or Formula II, or a combinationtherapy comprising a compound of Formula I, Formula IA, Formula IB, orFormula II is administered to an elderly human.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present disclosure areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentdisclosure. The examples do not limit the claimed disclosure. Based onthe present disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present disclosure.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties. However, where apatent, patent application, or publication containing expressdefinitions is incorporated by reference, those express definitionsshould be understood to apply to the incorporated patent, patentapplication, or publication in which they are found, and not to theremainder of the text of this application, in particular the claims ofthis application.

In some embodiments, the compound of Formula II is:

Compound No. STRUCTURE 1

2

3

4

5

6

71

77

76

107

111

126

133

137

139

141

143

145

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.

In some embodiments, compounds of Formula I, II, IA, IB or analogsthereof are:

7

8

10

11

12

14

16

17

18

19

20

21

22

27

28

29

30

31

32

33

34

35

36

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38

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41

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44

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46

47

48

49

50

51

54

61

63

65

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68

69

72

73

78

81

82

83

84

85

86

88

91

93

94

95

96

98

100

103

104

106

108

110

114

115

116

117

120

121

122

123

124

125

128

129

130

131

132

134

136

138

140

142

144

9

13

15

23

24

25

26

40

52

53

55

56

57

58

59

60

62

64

67

70

74

75

79

80

87

89

90

92

97

99

101

102

105

109

112

113

118

119

135

127

or pharmaceutically acceptable salts, solvates, enantiomers,diastereomers, racemates or mixtures thereof.

The HPLC plots were recorded on a Zorbax Eclipse Plus C18 column (4.6×50mm×1.8 μm). The first mobile phase was 97.5% water: 2.5% acetonitrile:0.05% trifluoroacetic acid. The second mobile phase was 97.5%acetonitrile: 2.5% water: 0.05% trifluoroacetic acid.

¹HNMR were recorded at 300 MHz unless otherwise specified.

Antiviral and Cytotoxicity Assays

Cells Culture and Virus Strains:

Human foreskin fibroblast (HFF) cells were prepared from human foreskintissue. The tissue was incubated at in cell culture media consisting ofminimum essential media (MEM) with Earle's salts supplemented with 10%fetal bovine serum (FBS), and standard concentrations of L-glutamine,fungizone, and vancomycin. Tissue was then placed in phosphate bufferedsaline (PBS), minced, rinsed to remove the red blood cells, andresuspended in trypsin/EDTA solution. The tissue suspension wasincubated at 37° C. and gently agitated to disperse the cells, whichwere then collected by centrifugation. Cells were resuspended in mediaand placed in a tissue culture flask and incubated at 37° C. in ahumidified CO₂ incubator. The media was then replaced with fresh mediaand the cell growth was monitored daily until a confluent cell monolayerwas formed. The HFF cells were then expanded through serial passages instandard growth medium of MEM with Earl's salts supplemented with 10%FBS, L-glutamine, penicillin, and gentamycin. The cells were passagedroutinely and used for assays at or below passage 10.

Influenza Virus:

Influenza viruses were passaged in canine kidney cells to create workingstocks, which were used for the antiviral assays.

Antiviral Assays:

Each experiment that evaluated the antiviral activity of the compoundsincluded both positive and negative control compounds to ensure theperformance of each assay. Concurrent assessment of cytotoxicity wasperformed when possible at equivalent levels of compound exposure.Methods are presented on effective concentrations giving 50% reductionsin viral replication in vitro (EC₅₀), concentrations producing 50%reduction in cell viability (CC₅₀) and selectivity index (SI, calculatedas CC₅₀ divided by EC₅₀). When sufficient material was available,multiple assays were performed for each compound evaluation to obtainstatistical data.

Cytotoxicity Assays:

Every antiviral assay included a parallel cytotoxicity assay with thesame cells used for each virus, the same cell number, the same drugconcentrations, and the same incubation times to provide the same drugexposure. To ensure that the cytotoxicity of all compounds could becompared directly, a standard neutral red uptake cytotoxicity assay forall compounds in confluent HFF cells with a 7 d incubation period wasperformed.

Neutral Red-Uptake Cytotoxicity Assays:

Each compound was evaluated in a standard cytotoxicity assay by standardmethods. Briefly, HFF cells were seeded into tissue culture plates instandard tissue culture medium. After 24 h of incubation, medium wasreplaced with maintenance cell culture medium and compounds were addedand then 5-fold serial dilutions were then used to generate a series ofcompound concentrations. Assay plates were incubated for 7 d, andneutral red solution in PBS was added to each well and the platesincubated for 1 h. The stain was then removed, the plates rinsed withPBS and the dye internalized by viable cells was solubilized in PBSsupplemented with 50% ethanol and 1% glacial acetic acid. The opticaldensity was then determined and CC₅₀ values were interpolated from theexperimental data.

For all plaque-reduction assays in HFF cells, neutral red cytotoxicityassays were performed on a parallel set of 6-well plates containinguninfected HFF cells that received the same compound concentrations asused for the antiviral assays. The cytotoxicity plates were removed fromthe incubator on the same day as each antiviral assay and the cellmonolayer was stained with a neutral red solution in PBS. The dye wasthen removed, residual dye rinsed from the cells with PBS, and cellmonolayers were inspected visually for any signs of toxicity. The cellviability was determined using a Cell Proliferation Assay according tomanufacturer's instructions.

Cell Proliferation Assays:

The inhibition of HFF cell proliferation was used to refine estimates ofcytotoxicity for some compounds and was performed according to astandard procedure used in the laboratory. Cells were seeded at a lowdensity into plates using standard culture medium. After 24 h, themedium was aspirated, and a range of compound solutions in the growthmedium was prepared. The plates were incubated for 72 h at 37° C., andthe cells were then dislodged with trypsin and counted. Compoundconcentrations that reduced cell proliferation by 50% were interpolatedfrom experimental data.

Cytotoxicity in Lymphocyte Assays:

Cell viability in all assays with lymphocytes was assessed with aluminescent Cell Viability Assay. Briefly, assay plates were incubatedat ambient temperature for 30 min then luminescent reagent was added toeach well and the plates were mixed to lyse the cells. Plates were thenincubated for at ambient temperature and the luminescence was quantifiedon a luminometer. Standard methods were used to calculate drugconcentrations that inhibited the proliferation of cells by 50% (CC₅₀).

Mouse Norovirus (MNV) Assay:

RAW cells were incubated at 37° C. with 5% CO₂ in cell culture media.The mouse norovirus isolate used in this assay was isolated from a wildmouse.

Serial dilutions of test compounds in DMEM were added to tissueculture-treated plates. Cell suspension were added to the compounddilutions in the plate, and incubated at 37° C. with 5% CO₂ for 2 days.Each assay plate included uninfected cell controls and untreated viruscontrol wells. After 2 days of incubation, aqueous MTS Reagent (wasadded as directed by manufacturer in each well and incubated at 37° C.until the untreated-cell controls developed a 490 nm absorbance between1.1 and 1.8. The final absorbance readings were read, and software wasused to calculate the concentration that protected MNV-infected RAWcells by 50% compared to uninfected cell controls (EC50).

Human Norovirus Assays:

Antiviral activity against a human Norovirus (NoV) was assessed in a3-day assay using a stably-expressing human Norovirus replicon cellline, maintained as sub-confluent cultures. In some embodiments, 4 doses(10-fold or 3-fold steps), in triplicate can be used. Antiviral activitywas determined by blot hybridization analysis of intracellular NoV RNA(normalized to the level of cellular β-actin RNA in each culturesample). Cytotoxicity was assessed by neutral red dye uptake in culturesmaintained in parallel plates (Korba and Gerin, 1992, Antivir. Res.19:55).

EC₅₀, and CC₅₀ values were calculated by linear regression analysisusing data combined from all treated cultures (Korba & Gerin, 1992,Antivir. Res. 19:55; Okuse, et al., 2005, Antivir. Res. 65:23). Standarddeviations for EC₅₀ and EC₉₀ values were calculated from the standarderrors generated by the regression analyses. EC₅₀ and EC₉₀ are drugconcentrations at which a 2-fold, or a 10-fold depression ofintracellular NoV RNA (relative to the average levels in untreatedcultures), respectively, is observed. CC₅₀ is the drug concentration atwhich a 2-fold lower level of neutral red dye uptake (relative to theaverage levels in untreated cultures) is observed. The Selectivity index(S.I.) is calculated as CC₅₀/EC₅₀. Recombinant human interferon 2b (PBLlaboratories, Inc.) is used as an assay control.

BKV EC₅₀ in VERO Cells:

Tissue culture plates were seeded with Vero cells in DMEM containing 2%Hyclone Standard Fetal Bovine Serum and 1% Hyclone Penicillin andStreptomycin. Cells were inoculated with BKV. Serial dilutions of testcompounds were added to the cells and plates were incubated for 10 daysat 37° C. in 5% CO₂. After the 10-day incubation, supernatant was mixedwith lysis buffer. Each plate was incubated C. Supernatant BKV DNA wasmeasured by quantitative polymerase chain reaction (qPCR) using forwardand reverse BKV PCR primers, and a FAM-labeled probe. Absolutequantitation of viral copy number was performed using a standard curvewith dilutions of a BKV DNA amplicon containing sequences homologous tothe amplified fragment.

Plaque-Reduction Assays for HSV-1, HSV-2, VZV and HCMV:

Monolayers of cells were prepared in six-well plates and incubated at toallow the cells to reach confluency. Media was then aspirated from thewells and of virus was added to each of three wells to yield 20-30plaques in each well. The virus was allowed to adsorb to the cells for 1h and the plates were rocked gently every 15 min to redistribute themedia. Compounds were diluted in maintenance cell culture mediaconsisting of MEM with Earl's salts supplemented with 2% FBS,L-glutamine, penicillin, and gentamycin. Solutions were added toduplicate wells and the plates were incubated for various times,depending on the virus used. The plaque-reduction assay with HSV-1strain F was performed in a similar manner but with Vero cells infectedone day after plating. For HSV-1 and -2, the monolayers were stainedwith 1% crystal violet in 20% methanol and the unbound dye removed bywashing with dH₂0. For assays with HCMV and VZV, the cell monolayer wasstained with 1% Neutral Red solution for 4 h then the stain wasaspirated and the cells were washed with PBS. For all assays, plaqueswere enumerated using a stereomicroscope and the concentration ofcompound that reduced plaque formation by 50% (EC50) was interpolatedfrom the experimental data.

Plaque-Reduction Assays for MCMV:

Mouse embryo fibroblast cells were prepared from mouse embryos using aprocedure similar to that outlined above for HFF cells and suspended intissue culture media as described above and seeded into plates andincubated. The medium was aspirated and the cell monolayers wereinfected with MCMV. The infected cells were then incubated at 37° C. for1 h and the plates were rocked occasionally to ensure that the mediacovered the entire monolayer. Compounds were serially diluted in tissuemaintenance cell culture media described above and the solutions wereadded to the infected monolayers. Infected monolayers were incubated for7 d and then stained with Neutral Red solution as described above.Plaques were enumerated and EC₅₀ values were interpolated from theexperimental data by standard methods.

DNA Quantitation Assays for EBV and HHV-6B:

Assays for EBV were performed in Akata cells that were induced toundergo a lytic infection with goat anti-human IgG antibody by standardmethods. Experimental compounds were diluted in plates. Akata cells wereadded to the plates and incubated. For HHV-6, compounds were seriallydiluted then uninfected HSB-2 or Molt-3 cells were added to each well.Infection was initiated by adding HHV-6A infected HSB-2 cells, or HHV-6Binfected Molt-3 cells, and incubated for 7 d at 37° C. For all assays,denaturation buffer was added to each well to denature the DNA and analiquot was aspirated through a nylon membrane. The membranes were thenallowed to dry before equilibration in DIG. Specific digoxigenin(DIG)-labeled probes were prepared for each virus according to themanufacturer's protocol. Detection of specifically bound DIG probe wasperformed with anti-DIG antibody using the manufacturer's protocol. Animage of the photographic film was captured and quantified and compoundconcentrations sufficient to reduce the accumulation of viral DNA by 50%(EC50), were interpolated from the experimental data.

DNA Quantitation Assays for HHV-8:

Test compounds were diluted in duplicate wells. BCBL-1 cells wereinduced to undergo a lytic infection by the addition of phorbol12-myristate 13-acetate cells were added to each well in the plate.Cells were incubated for 7 d at 37° C. in a humidified CO₂ incubatorthen total DNA was prepared. Viral DNA was quantified by real time PCRCompound concentrations sufficient to reduce genome copy number by 50%were calculated from experimental data.

Cell-Based Assays for Flu:

For dose-response curves, individual drugs were added to MDCK cells in96-well microplates. Untreated wells of infected cells (virus controls)and uninfected cells (cell controls) were included on each test plate.At three days post-infection, the virus control wells exhibited 100%cytopathology. The extent of viral cytopathology in each well wasdetermined microscopically by inspection and by staining with neutralred (NR). Briefly, the cells were stained with NR diluted in MEM todetermine cell viability. Two hours later the plates were processed forquantification of NR uptake into viable cells. The amount of NR taken upby cells was determined spectrophotometrically.

qPCR Assays for BKV and JCV:

Primary assays for BK virus were performed in 96-well plates containingmonolayers of HFF cells. Compound dilutions were prepared in platescontaining cells which were subsequently infected at with the Gardnerstrain of BK virus. After a 7 d incubation, total DNA was prepared witha purification kit and genome copy number was quantified by real timePCR. Compounds that were positive in this assay were confirmed in asimilar assay in 96-well plates with the compounds added 1h followinginfection to identify compounds that inhibit early stages of replicationincluding adsorption and penetration. Genome copy number was determinedby methods described above.

Primary evaluation of compounds against JC virus were also performed bymethods similar to those for BK virus primary assays. Secondary assaysagainst JCV were performed in COST cells by methods similar to those forBK virus to identify compounds that inhibited adsorption or penetrationof the virus.

Hepatitis C Virus Assay:

Luciferase reporter (Replicon)/CytoTox-1 (Toxicity). Compounds werescreened for anti-HCV activity using a luciferase (Luc) reporter geneendpoint in the HCV primary assay. The Luc reporter was used as anindirect measure of HCV replication as its activity is directlyproportional to HCV RNA levels. Assessment of cytotoxicity is conductedin parallel. Drug stocks were prepared in DMSO unless otherwisespecified and were diluted with tissue culture medium to the desiredhigh-test concentration. For each assay, the compounds were then furtherdiluted in tissue culture medium as required. After incubation, thecells were processed to derive, where applicable, EC50 and EC90(compound concentration reducing replicon replication by 50% and 90%respectively). CC50 (concentration decreasing cell viability by 50%) andSI50 (CC50/EC50) values were determined and reported. Anti-HCV activitywas assessed with the replicon (genotype 1b or 2a) or HCVccvirus-derived Luc activity as readout; whereas the cytotoxicconcentrations of drug reducing cell numbers was assessed by theCytoTox-1 cell proliferation assay according to manufacturer's protocol.Recombinant interferon alpha was used as the positive control drug tovalidate assay performance.

Assays for Influenza virus, RSV, and SARS CoV were Cytopathiceffect/Toxicity-based assay using CellTiter-Glo. The antiviralcytoprotection assays examine the effects of compounds at designateddose-response concentrations in specific cell types to test the efficacyof the compounds in preventing the virus-induced cytopathic effect.Ribavirin was included as a positive control drug for influenza and RSV,while calpain IV inhibitor was used for SARS antiviral assays.Subconfluent cultures of cells were plated into 96-well plates for theanalysis of cell viability (cytotoxicity) and antiviral activity (CPE).For the standard assay, drugs were added to the cells 24 hours later.The CPE wells also received 100 tissue culture infectious doses (100TCID50s) of titered virus. 72 hours later the cell viability wasdetermined.

Measurement of viral-induced CPE was based on quantitation of ATP, anindicator of metabolically active cells. The CPE assay employed acommercially available Luminescent Cell Viability Kit, and was areliable method for determining cytotoxicity and cell proliferation inculture. The procedure involved adding the single reagent directly topreviously cultured, subconfluent cells in media. This induced celllysis and the production of a bioluminescent signal (half-life greaterthan 5 hours, depending on the cell type) that was proportional to theamount of ATP present (which is a biomarker for viability).

Assays for Dengue (DENV), West Nile Virus (WNV), Yellow Fever Virus(YFV), Rift Valley Fever Virus (RVFV), Venezuelan Equine EncephalitisVirus (VEEV):

Primary cytopathic effect (CPE) reduction assay. Four-concentration CPEinhibition assays were performed. Confluent or near-confluent cellculture monolayers in microplates were prepared. Cells were maintainedin MEM or DMEM supplemented with FBS as required for each cell line. Forantiviral assays the same medium is used but with FBS reduced to 2% orless and supplemented with gentamicin. The test compound was prepared atdifferent concentrations, u. The virus control and cell control wellswere on every microplate. In parallel, a known active drug was tested asa positive control drug using the same method as was applied for testcompounds. The positive control was tested with each test run. The assaywas set up by first removing growth media from the 96-well plates ofcells. Then the test compound was applied to wells. Virus was placed inthose wells designated for virus infection. Medium devoid of virus wasplaced in toxicity control wells and cell control wells. Virus controlwells was treated similarly with virus. Plates were incubated at 37° C.with 5% CO₂ until maximum CPE is observed in virus control wells. Theplates were then stained with neutral red for approximately two hours at37° C. in a 5% CO₂ incubator. The neutral red medium was removed bycomplete aspiration, and the cells can be rinsed with phosphate bufferedsolution (PBS) to remove residual dye. The PBS is completely removed andthe incorporated neutral red was eluted with buffer. The dye content ineach well is quantified using a spectrophotometer. The dye content ineach set of wells was converted to a percentage of dye present inuntreated control wells using a Microsoft Excel computer-basedspreadsheet. The 50% effective (EC50) concentrations and 50% cytotoxic(CC50) concentrations were then calculated by linear regressionanalysis. The quotient of CC50 divided by EC50 gives the selectivityindex (SI) value.

Assays for Adenovirus (AdV), Measles (MEV), Poliovirus (POV) andEnterovirus (ENTV):

The primary screen was a cytopathic effect (CPE) reduction assay.Briefly, cultures of cells were infected with virus in the presence oftest compounds and incubated for 4-7 days (depending on the specificvirus/cells). Each virus was pre-titered such that control wellsexhibited approximately 95% loss of cell viability due to virusreplication. Therefore, antiviral effect, or cytoprotection, wasobserved when compounds prevent virus replication. Each assay platecontained cell control wells (cells only), virus control wells (cellsplus virus), compound toxicity control wells (cells plus compound only),compound colorimetric control wells (compound only, no cells or virus),as well as experimental wells (compound plus cells plus virus).Cytoprotection and compound cytotoxicity were assessed by MTS dyereduction. The percent reduction in viral CPE (antiviral activity) andpercent cell viability (cytotoxicity) were determined and reported.

Assays for Vaccinia Virus (VACV):

The primary assay was a cytopathic effect (CPE) reduction assay. Lowpassage HFF cells were trypsinized, counted, and seeded into tissueculture plates. The cells were then incubated for 24 h at 37° C. Themedia was then removed and MEM containing 2% FBS was added to all butthe first row. In the first row, media containing the experimental drug(e.g., Compound 1) was added in triplicate wells. Media alone was addedto both cell and virus control wells. The drug in the first row of wellswas then diluted serially 1:5 throughout the remaining wells. The plateswere then incubated for 60 minutes and a virus suspension was added toeach well, excluding cell control wells which received MEM. The plateswere then incubated at 37° C. in a CO₂ incubator. After the incubationperiod, media was aspirated and the cells stained with crystal violet informalin for 4h. The stain was then removed and the plates were rinseduntil all excess stain was removed. The plates were allowed to dry for24 h and the amount of CPE in each row determined. EC₅₀ and CC₅₀ valueswere determined by comparing drug treated and untreated cells using acomputer program.

As set forth in Example 1, below, compound 1 has activity against mousenorovirus in vitro. In some embodiments, compound 1 has an EC₅₀ value ofabout 2.1 and a CC₅₀ value of about 114 against mouse norovirus.Additionally, compound 1 has activity against a wide array of DNA andRNA viruses.

As set forth in Example 2, below, compound 1 has activity against mousenorovirus in vivo. As set forth in Example 2, compound 1 was able toreduce the viral load in mice infected with murine norovirus in adose-dependent manner. The results are given in FIG. 1A and FIG. 1B,which show the reduction of viral titer in the feces and the tissue,respectively, of mice in Study 1. Additionally, FIGS. 2A and 2B show thereduction of viral titer in the feces and tissue, respectively, of micein Study 2. FIGS. 3A and 3B show the reduction in the viral titer in thetissue and feces, respectively, of the mice in Study 1. The results areshown on a linear scale. Mice were treated with 30/mg/kg/day, or 100mg/kg/day, or 300 mg/kg/day of compound 1 in Study 1. Mice were treatedwith 150/mg/kg/day, or 300 mg/kg/day of compound 1 in Study 2.

Example 3 demonstrates that compound 1 is capable of inhibitingnorovirus polymerase in vitro. Without wishing to be bound by theory, itis proposed that compound 1 can protect against and treat norovirus byinhibiting norovirus polymerase.

As set forth in Example 4, compound 1 is converted to a triphosphate invitro. As shown, when cells were incubated with compound 1, thecorresponding triphosphate (i.e., compound 1-TP) was produced. The levelof Compound 1-TP was 12 to 23-fold higher than compound 1 after theincubation period.

Example 5, below, demonstrates that compound 1 is more effective atinhibiting mouse norovirus than compound 2, or than 2′-C-methylcytidinetriphosphate. A comparison with DMSO as a control is given forreference. The experiment was conducted in duplicate and the results areshown in FIG. 4 (first duplicate) and FIG. 5 (second duplicate). FIG. 6shows an overlay of the results of the first and second duplicate of theexperiment. As shown in FIGS. 4, 5 and 6, “A” is DMSO, “B” is Compound2, “C” is 2′-C-methylcytidine triphosphate (2′CmeC TP) and “D” isCompound 1. FIGS. 4-6 demonstrate that the viral titer increased almosttwo orders of magnitude when treated with only DMSO or 2′CmeC TP.However, the viral titer increased less than one order of magnitude inthe presence of compound 1.

Example 6 shows a comparison with compound 1 and other compounds ofFormula II (Table 7) and analogs thereof. Without wishing to be bound bytheory, compounds that are even slightly different from the structure ofFormula I can have substantially reduced activity in vitro. For example,compound 7 has a hydroxy group in place of the aryl amine and has an hasan EC₅₀ value >38 μM; compound 35 shows an analog of compound 1 lackingthe aryl amine group, and has an EC₅₀ value >100 μM. Additionally,compound 11 has a cyano group in place of the aryl amide and has an EC₅₀value >121 μM. Similarly, compounds 36 and 37 show methyl-substitutedamines and have EC₅₀ values >100 μM. Additional comparisons will beunderstood by one of skill in the art.

FIG. 7a shows an HPLC plot of compound 1.

Peak Ret. Width Area Height No. Time Type (min) (mAu*s) (mAU) Area % 13.649 MM 0.0449 2712.41235 1007.2742 98.2495 2 4.928 MM 0.0511 48.3275515.75879 1.7505Without wishing to be bound by theory, peak no. 2 was identified asbenzoic acid.

FIG. 7b shows an HPLC plot of compound 1 after slurrying 3 hours at roomtemperature

Peak Ret. Width Area Height No. Time Type (min) (mAu*s) (mAU) Area % 13.641 MM 0.0448 2621.76538 975.76003 99.9122 2 4.923 MM 0.0519 2.304310.739979 0.0878

FIG. 7c shows an HPLC plot of compound 1 after slurrying 3 hours at 50°C.

Peak Ret. Width Area Height No. Time Type (min) (mAu*s) (mAU) Area % 13.609 MM 0.0446 2541.75464 949.26025 99.9317 2 4.888 MM 0.473 1.7375950.0612609 0.683

FIG. 7d shows an HPLC plot of compound 1 after slurrying 24 hours atabout room temperature.

Peak Ret. Width Area Height No. Time Type (min) (mAu*s) (mAU) Area % 13.577 MM 0.0452 2749.37183 1013.14984 99.9256 2 4.888 MM 0.0500 2.046370.0682284 0.0744

FIG. 8a shows a ¹HNMR spectrum of compound 1 from about −2 to about 14ppm.

FIG. 8b shows a ¹HNMR spectrum of compound 1 from about 2 to about 9ppm.

FIG. 8c shows a ¹HNMR spectrum of compound 1 from about 0 to about 9ppm.

EXAMPLES

General Procedures

The HPLC plots were recorded on a Zorbax Eclipse Plus C18 column (4.6×50mm×1.8 μm). The first mobile phase was 97.5% water: 2.5% acetonitrile:0.05% trifluoroacetic acid. The second mobile phase was 97.5%acetonitrile: 2.5% water: 0.05% trifluoroacetic acid.

¹HMNR were recorded at 500 MHz unless otherwise specified.

Example 1—Antiviral and Cytotoxicity Assays

Cells Culture and Virus Strains:

Human foreskin fibroblast (HFF) cells were prepared from human foreskintissue obtained from the University of Alabama at Birmingham tissueprocurement facility with approval from its IRB. The tissue wasincubated at 4° C. for 4 h in cell culture media consisting of minimumessential media (MEM) with Earle's salts supplemented with 10% fetalbovine serum (FBS) (Hyclone, Inc. Logan Utah), and standardconcentrations of L-glutamine, fungizone, and vancomycin. Tissue wasthen placed in phosphate buffered saline (PBS), minced, rinsed to removethe red blood cells, and resuspended in trypsin/EDTA solution. Thetissue suspension was incubated at 37° C. and gently agitated todisperse the cells, which were then collected by centrifugation. Cellswere resuspended in 4 mL media and placed in a 25 cm² tissue cultureflask and incubated at 37° C. in a humidified CO₂ incubator for 24 h.The media was then replaced with fresh media and the cell growth wasmonitored daily until a confluent cell monolayer was formed. The HFFcells were then expanded through serial passages in standard growthmedium of MEM with Earl's salts supplemented with 10% FBS, L-glutamine,penicillin, and gentamycin. The cells were passaged routinely and usedfor assays at or below passage 10.

Akata cells latently infected with EBV were obtained from John Sixbey(Louisiana State University, Baton Rouge, La.). The GS strain of HHV-6Awas obtained through the NIH AIDS Research and Reference ReagentProgram. HSB-2 cells and BCBL-1 cells were obtained through the NIH AIDSResearch and Reference Reagent Program, Division of AIDS, NIAID, NIH.Molt-3 cells were obtained from Scott Schmid at the Centers for DiseaseControl and Prevention, Atlanta, Ga. All lymphocyte cultures weremaintained routinely in RPMI 1640 (Mediatech, Inc., Herndon, Va.) with10% FBS, L-glutamine and antibiotics and passaged twice a week. Verocells were obtained from American Type Culture Collection (ATCC,Manassas, Va.), and were maintained in standard growth medium of MEMwith Earl's salts supplemented with 10% FBS, L-glutamine, penicillin,and streptomycin.

Influenza Virus:

Influenza A/New Caledonia/20/99 (H1N1) and A/Sydney/05/97 (H3N₂) viruswere provided by the Centers for Disease Control and Prevention(Atlanta, Ga.). The viruses were passaged in Madin-Darby canine kidney(MDCK) cells (American Type Culture Collection, Manassas, Va.) to createworking stocks, which were used for the antiviral assays.

The E-377 and DM2.1 strains of HSV-1 as well as the MS and 13078 strainsof HSV-2 were used. HSV-1 strain F was obtained from ATCC. The HCMVstrains, AD169 and Merlin were obtained from the American Type CultureCollection (ATCC, Manassas, Va.) and C8805/37-1-1 and 759RD100 were agift of Karen Biron. VZV, strain Ellen was obtained from the ATCC. TheZ29 strain of HHV-6B was a gift of Scott Schmid at the Centers forDisease Control and Prevention, Atlanta Ga. HHV-8 was obtained aslatently infected BCBL-1 cells through the NIH AIDS Research andReference Reagent Program.

Antiviral Assays:

Each experiment that evaluated the antiviral activity of the compoundsincluded both positive and negative control compounds to ensure theperformance of each assay. Concurrent assessment of cytotoxicity wasperformed when possible at equivalent levels of compound exposure.Methods are presented following the tabulated data on effectiveconcentrations giving 50% reductions in viral replication in vitro(EC50), concentrations producing 50% reduction in cell viability (CC50)and selectivity index (SI, calculated as CC50 divided by EC50). Whensufficient material was available, multiple assays were performed foreach compound evaluation to obtain statistical data.

Cytotoxicity Assays:

Every antiviral assay included a parallel cytotoxicity assay with thesame cells used for each virus, the same cell number, the same drugconcentrations, and the same incubation times to provide the same drugexposure. To ensure that the cytotoxicity of all compounds could becompared directly, a standard neutral red uptake cytotoxicity assay forall compounds in confluent HFF cells with a 7 d incubation period wasperformed.

Neutral Red-Uptake Cytotoxicity Assays:

Each compound was evaluated in a standard cytotoxicity assay by standardmethods. Briefly, HFF cells were seeded into 96-well tissue cultureplates at a 2.5×10⁴ cells/well in standard tissue culture medium. After24 h of incubation, medium was replaced with maintenance cell culturemedium and compounds were added to the first row and then 5-fold serialdilutions were then used to generate a series of compound concentrationswith a maximum of 300 μM. Assay plates were incubated for 7 d, and 100μL of a 0.66 mg/mL neutral red solution in PBS was added to each welland the plates incubated for 1 h. The stain was then removed, the platesrinsed with PBS and the dye internalized by viable cells was solubilizedin PBS supplemented with 50% ethanol and 1% glacial acetic acid. Theoptical density was then determined at 550 nm and CC50 values wereinterpolated from the experimental data.

For all plaque-reduction assays in HFF cells, neutral red cytotoxicityassays were performed on a parallel set of 6-well plates containinguninfected HFF cells that received the same compound concentrations asused for the antiviral assays. The cytotoxicity plates were removed fromthe incubator on the same day as each antiviral assay and the cellmonolayer was stained for 6 h with 2 mL of a neutral red solution at aconcentration of 0.165 mg/mL in PBS. The dye was then removed, residualdye rinsed from the cells with PBS, and cell monolayers were inspectedvisually for any signs of toxicity. Cytotoxicity assay with Vero cellswas performed with drug concentrations ranging from 1 μM to 1 mM. Thecell viability was determined using CellTiter 96® Aqueous One SolutionCell Proliferation Assay (Promega) according to manufacturer'sinstructions.

Cell Proliferation Assays:

The inhibition of HFF cell proliferation was used to refine estimates ofcytotoxicity for some compounds and was performed according to astandard procedure used in the laboratory. Cells were seeded at a lowdensity into six-well plates using 2.5×10⁴ cells/well and standardculture medium. After 24 h, the medium was aspirated, and a range ofcompound solutions in the growth medium was prepared starting at 300 μMand added to duplicate wells. The plates were incubated for 72 h at 37°C., and the cells were then dislodged with trypsin and counted on aBeckman Coulter Counter. Compound concentrations that reduced cellproliferation by 50% were interpolated from experimental data.

Cytotoxicity in Lymphocyte Assays:

Cell viability in all assays with lymphocytes was assessed with theCellTiter-Glo Luminescent Cell Viability Assay (Promega). Briefly, assayplates were incubated at ambient temperature for 30 min then 50 μL ofCellTiter-Glo reagent was added to each well and the plates were mixedfor 2 min on an orbital shaker to lyse the cells. Plates were thenincubated for an additional 10 min at ambient temperature and theluminescence was quantified on a luminometer. Standard methods were usedto calculate drug concentrations that inhibited the proliferation ofAkata, HSB-2, BCLB-1, or Molt-3 cells by 50% (CC₅₀).

TABLE 1 Activity of Compound 1 against mouse norovirus in RAW cells MNVRAW Cells Compound Mean EC₅₀, μM Mean CC₅₀, μM SI Compound 1 2.0 +/− 0.8114.0 +/− 12.5 57.0 (n = 21) (n = 4) Compound 1 2.1 (n = 33) 114 54(2′-C-Methyl Cytidine) 3.2 +/− 1.5 34.3 +/− 9.3 10.7 (n = 20) (n = 8)

TABLE 2 Activity of Compound 1 against various DNA and RNA viruses VirusEC₅₀ (μM) CC₅₀ (μM) SI Cell line DNA Viruses AdV >100 >100 ~1 A549BKV >60 >60 ~1 HFF EBV 7.15 >60 >8.4 Akata HCMV >60 >60 ~1 HFFMCMV >100 >100 ~1 MEF VZV >60 >60 ~1 HFF HSV-2 >60 >60 ~1 HFF HHV-6B39.66 44.71 1.13 MOLT-3 HHV-8 >12.00 46.75 <3.9 BCBL-1 VACV 56.16 >60 ~1HFF JCV 0.46 >60 >130 293TT MEV >100 >100 ~1 Vero76 RNA VirusesDENV-2 >100 >100 ~1 Vero76 ENTV-71 >100 >100 ~1 Vero76 HCV 18.4 >20 >1.1Huh 7 Flu (H1N1) >100 >100 ~1 MDCK POV-3 >100 >100 ~1 Vero76 RSV 31 331.1 MA-104 SARS 37 >100 >2.7 Vero76 RVFV >100 >100 ~1 Vero76VEEV >100 >100 ~1 Vero76 YFV >100 >100 ~1 Vero WNV >100 >100 ~1 Vero76

Mouse Norovirus (MNV) Assay:

RAW cells (Mouse Macrophage, ATCC TIV-71) were received from AmericanType Culture Collection (ATCC). The cells were incubated at 37° C. with5% CO₂ in cell culture media consisting of Dulbecco's minimum essentialmedia (DMEM) (ATCC) supplemented with 10% fetal bovine serum (FBS)(Hyclone, Inc., Logan Utah), 100 U/mL penicillin and 100 ug/mLstreptomycin (Hyclone), 1% MEM NEAA (Gibco), 1% GlutaMAX (Gibco), and 1%HEPES (Hyclone). The mouse norovirus isolate used in this assay wasisolated from a wild mouse, cell-culture adapted, plaque purified, andgenetic sequence confirmed by Chimerix, Inc.

100 μL of serial dilutions of test compounds in DMEM were added toCostar 96-well tissue culture-treated plates. 100 μL of cell suspensioncontaining 50,000 RAW cells/well and MNV (MOI=0.0005) were added to thecompound dilutions in the 96-well plate, and incubated at 37° C. with 5%CO₂ for 2 days. Each assay plate included uninfected cell controls anduntreated virus control wells. After 2 days of incubation, the untreatedvirus control wells showed 100% CPE. After 2 days of incubation, 40 μLof Cell Titer 96® Aqueous MTS Reagent (Promega, G111) was added asdirected by manufacturer to the 200 μL media in each well and incubatedat 37° C. until the untreated-cell controls developed a 490 nmabsorbance between 1.1 and 1.8. The final absorbance readings were readusing a BioTek Synergy 2, and Gen 5 software (BioTek Instruments, Inc.)was used to calculate the concentration that protected MNV-infected RAWcells by 50% compared to uninfected cell controls (EC50).

Human Norovirus Assays:

Antiviral activity against a human Norovirus (NoV) was assessed in a3-day assay using the stably-expressing human Norovirus replicon cellline, HG23 (genogroup I, genomic length; parental cell line, HuH7)(Chang, et al., 2006, Virol. 353:463) maintained as sub-confluentcultures on 96-well plates. Typically, 4 doses (10-fold or 3-foldsteps), in triplicate are used. Antiviral activity was determined byblot hybridization analysis of intracellular NoV RNA (normalized to thelevel of cellular β-actin RNA in each culture sample). Cytotoxicity wasassessed by neutral red dye uptake in cultures maintained in parallelplates (Korba and Gerin, 1992, Antivir. Res. 19:55).

EC₅₀, and CC₅₀ values are calculated by linear regression analysis (MSEXCEL®, QuattroPro®) using data combined from all treated cultures(Korba & Gerin, 1992, Antivir. Res. 19:55; Okuse, et al., 2005, Antivir.Res. 65:23). Standard deviations for EC₅₀ and EC90 values werecalculated from the standard errors generated by the regressionanalyses. EC₅₀ and EC₉₀ are drug concentrations at which a 2-fold, or a10-fold depression of intracellular NoV RNA (relative to the averagelevels in untreated cultures), respectively, was observed. CC₅₀ is thedrug concentration at which a 2-fold lower level of neutral red dyeuptake (relative to the average levels in untreated cultures) isobserved. The Selectivity index (S.I.) was calculated as CC₅₀/EC₅₀.Recombinant human interferon 2b (PBL laboratories, Inc.) is used as anassay control.

BKV EC₅₀ in VERO Cells:

Costar 96-well tissue culture plates were seeded with 10,000 Verocells/well in DMEM containing 2% Hyclone Standard Fetal Bovine Serum(FBS, Cat SH30088.03) and 1% Hyclone Penicillin and Streptomycin. Outerwells were not used to minimize the edge-effect produced by extendedincubations. Cells were inoculated with 115 BKV DNA copies/cell (ATCC,Gardner strain). Serial dilutions of test compounds were added to thecells and plates were incubated for 10 days at 37° C. in 5% CO₂. Afterthe 10-day incubation, 50 μL supernatant was mixed with 50 μL 2× lysisbuffer that provided a final concentration 0.5 mg/mL protease K, 50 mMKCl, 10 mM Tris-Cl pH 8.0, 2.5 mM MgCl2, 0.45% IGEPAL, and 0.45%Tween-20 dissolved in DEPC-treated water. Each plate was incubated for 2hours at 55° C. Supernatant BKV DNA was measured by quantitativepolymerase chain reaction (qPCR) using forward and reverse BKV PCRprimers, and a FAM-labeled probe. Absolute quantitation of viral copynumber was performed using a standard curve with dilutions of a BKV DNAamplicon containing sequences homologous to the amplified fragment. Thefollowing qPCR amplification conditions were used: 1 cycle at 95° C. for10 minutes, followed by 45 cycles of 95° C. for 15 seconds and 60° C.for 60 seconds. The qPCR reactions were performed using an AppliedBiosystems 7500 real Time PCR System. Gen 5 software (BioTekInstruments, Inc.) was used to calculate the concentration whichinhibited the viral DNA levels of BKV-infected Vero cells by 50% (EC50).

Plaque-Reduction Assays for HSV-1, HSV-2, VZV and HCMV:

Monolayers of HFF cells were prepared in six-well plates and incubatedat 37° C. for 2 d to allow the cells to reach confluency. Media was thenaspirated from the wells and 0.2 mL of virus was added to each of threewells to yield 20-30 plaques in each well. The virus was allowed toadsorb to the cells for 1 h and the plates were rocked gently every 15min to redistribute the media. Compounds were diluted in maintenancecell culture media consisting of MEM with Earl's salts supplemented with2% FBS, L-glutamine, penicillin, and gentamycin. Solutions ranging from300 μM to 0.1 μM were added to duplicate wells and the plates wereincubated for various times, depending on the virus used. Theplaque-reduction assay with HSV-1 strain F was performed in a similarmanner but with Vero cells infected one day after plating. Final FBSconcentration in this assay was 5%. For HSV-1 and -2, the monolayerswere stained with 1% crystal violet in 20% methanol and the unbound dyeremoved by washing with dH₂0. For assays with HCMV and VZV, the cellmonolayer was stained with 1% Neutral Red solution for 4 h then thestain was aspirated and the cells were washed with PBS. For all assays,plaques were enumerated using a stereomicroscope and the concentrationof compound that reduced plaque formation by 50% (EC50) was interpolatedfrom the experimental data.

Plaque-Reduction Assays for MCMV:

Mouse embryo fibroblast cells were prepared from mouse embryos using aprocedure similar to that outlined above for HFF cells and suspended intissue culture media as described above and seeded into 12 well platesand incubated at 37° C. for 24 h. The medium was aspirated and the cellmonolayers were infected with the Smith strain of MCMV in a final volumeof 0.2 mL in each of triplicate wells. The infected cells were thenincubated at 37° C. for 1 h and the plates were rocked occasionally toensure that the media covered the entire monolayer. Compounds wereserially diluted 1:5 in tissue maintenance cell culture media describedabove and the solutions were added to the infected monolayers. Infectedmonolayers were incubated for 7 d and then stained with 2 mL of a 1%Neutral Red solution as described above. Plaques were enumerated andEC₅₀ values were interpolated from the experimental data by standardmethods.

DNA Quantitation Assays for EBV and HHV-6B:

Assays for EBV were performed in Akata cells that were induced toundergo a lytic infection with 50 μg/mL of a goat anti-human IgGantibody by standard methods. Experimental compounds were diluted inround bottom 96-well plates to yield concentrations ranging from 20 to0.016 μM. Akata cells were added to the plates at a concentration of4×10⁴ cells per well and incubated for 72 h. For HHV-6, compounds wereserially diluted in 96-well plates then 1×10⁴ uninfected HSB-2 or Molt-3cells were added to each well. Infection was initiated by adding HHV-6Ainfected HSB-2 cells, or HHV-6B infected Molt-3 cells, at a ratio ofapproximately 1 infected cell for every 10 uninfected HSB-2 cells orMolt-3 cells respectively and incubated for 7 d at 37° C. For allassays, 100 μL of denaturation buffer (1.2M NaOH, 4.5M 80 NaCl) wasadded to each well to denature the DNA and a 50 μL aliquot was aspiratedthrough an Immobilon nylon membrane (Millipore, Bedford, Mass.) using aBiodot apparatus (Bio-Rad, Hercules, Calif.). The membranes were thenallowed to dry before equilibration in DIG Easy Hyb (Roche Diagnostics,Indianapolis, Ind.) at 56° C. for 30 min. Specific digoxigenin(DIG)-labeled probes were prepared for each virus according to themanufacturer's protocol (Roche Diagnostics). For EBV, primers 5′-CCC AGGAGT CCC AGT AGT CA-3′ (SEQ ID No. 1) and 5′-CAG TTC CTC GCC TTA GGTTG-3′ (SEQ ID No. 2) amplified a fragment corresponding to coordinates96802-97234 in EBV genome (AJ507799). A specific HHV-6 DIG labeled probewas prepared using primers 5′-CCT TGA TCA TTC GAC CGT TT-3′ (SEQ ID No.3) and 5′-TGG GAT TGG GAT TAG AGC TG-3′ (SEQ ID No. 4) to amplify asegment of ORF2 (coordinates 37820-38418 in X₈₃₄₁₃). Membranes with EBVDNA were hybridized overnight at 56° C. followed by sequential washes in0.2×SSC with 0.1% SDS and 0.1×SSC with 0.1% SDS at the same temperature.For HHV-6A and HHV-6B blots, the probe was allowed to hybridizeovernight at 42° C. and the blots were rinsed at the same temperaturewith 0.2×SSC with 0.1% SDS and 0.1×SSC with 0.1% SDS. Detection ofspecifically bound DIG probe was performed with anti-DIG antibody usingthe manufacturer's protocol (Roche Diagnostics). An image of thephotographic film was captured and quantified with QuantityOne software(Bio-Rad) and compound concentrations sufficient to reduce theaccumulation of viral DNA by 50% (EC50), were interpolated from theexperimental data.

DNA Quantitation Assays for HHV-8:

Test compounds were diluted in duplicate wells of a 96-well plate withthe highest final concentration of 20 μM. BCBL-1 cells were induced toundergo a lytic infection by the addition of phorbol 12-myristate13-acetate (Promega, Madison Wis.) at a final concentration of 100 ng/mLand 2×10⁴ cells were added to each well in the plate. Cells wereincubated for 7 d at 37° C. in a humidified CO₂ incubator then total DNAwas prepared with a Wizard SV 96 well purification kit (Promega). ViralDNA was quantified by real time PCR using forward primer 5′-TTC CCC AGATAC ACG ACA GAA TC-3′ (SEQ ID No. 5), reverse primer 5′-CGG AGC GCA GGCTAC CT-3′ (SEQ ID No. 6), and probe 5′-(FAM) CCT ACG TGT TCG TCG AC(TAMRA)-3′ (SEQ ID No. 7). Plasmid pMP218 containing a DNA sequencescorresponding to nucleotides 14120-14182 (AF148805.2) was used toprovide absolute quantification of viral DNA. Compound concentrationssufficient to reduce genome copy number by 50% were calculated fromexperimental data.

Cell-Based Assays for Flu:

For dose-response curves, individual drugs were added to MDCK cells in96-well microplates (8×10⁴ cells/well) using three wells for eachconcentration used. The compounds were added at the followingconcentrations: oseltamivir carboxylate at 0, 0.000032, 0.0001, 0.00032,0.001, 0.0032, 0.01, 0.032, 0.1, 1.0, 10.0 and 100 μg/mL; amantadine andribavirin at 0, 0.001, 0.0032, 0.01, 0.032, 0.1, 0.32, 1, 3.2, 10, 32and 100 m/mL. Untreated wells of infected cells (virus controls) anduninfected cells (cell controls) were included on each test plate. Atthree days post-infection, the virus control wells exhibited 100%cytopathology. The extent of viral cytopathology in each well wasdetermined microscopically by inspection and by staining with neutralred (NR). Briefly, the cells were stained with 0.011% NR diluted in MEMto determine cell viability. Two hours later the plates were processedfor quantification of NR uptake into viable cells. The amount of NRtaken up by cells was determined spectrophotometrically.

qPCR Assays for BKV and JCV:

Primary assays for BK virus were performed in 96-well plates containingmonolayers of HFF cells. Compound dilutions were prepared in platescontaining cells which were subsequently infected at with the Gardnerstrain of BK virus. After a 7 d incubation, total DNA was prepared witha Wizard SV 96 well purification kit and genome copy number wasquantified by real time PCR using the primers 5′-AGT GGA TGG GCA GCC TATGTA-3′ (SEQ ID No. 8), 5′-TCA TAT CTG GGT CCC CTG GA-3′ (SEQ ID No. 9)and probe 5′-6-FAM AGG TAG AAG AGG TTA GGG TGT TTG ATG GCA CAGTAIVIRA-3′ (SEQ ID No. 10). Plasmid pMP526 served as the DNA standardfor quantitation purposes. Compounds that were positive in this assaywere confirmed in a similar assay in 96-well plates with the compoundsadded 1h following infection to identify compounds that inhibit earlystages of replication including adsorption and penetration. Genome copynumber was determined by methods described above.

Primary evaluation of compounds against JC virus were also performed bymethods similar to those for BK virus primary assays but were done in293TT cells and utilized the 1-4 strain of JCV in 293TT cells. Viral DNAwas quantified using primers 5′-CTG GTC ATG TGG ATG CTG TCA-3′ (SEQ IDNo. 11) and 5′-GCC AGC AGG CTG TTG ATA CTG-3′ (SEQ ID No. 12) and probe5′-6-FAM-CCC TTT GTT TGG CTG CT-TAMRA-3′ (SEQ ID No. 13) together withthe plasmid pMP508 to provide a standard curve for absolutequantification. Secondary assays against JCV were performed in COS7cells by methods similar to those for BK virus to identify compoundsthat inhibited adsorption or penetration of the virus.

Hepatitis C Virus Assay:

Luciferase reporter (Replicon)/CytoTox-1 (Toxicity). Compounds werescreened for anti-HCV activity using a luciferase (Luc) reporter geneendpoint in the HCV primary assay. The Luc reporter was used as anindirect measure of HCV replication as its activity was directlyproportional to HCV RNA levels. Assessment of cytotoxicity is conductedin parallel. Drug stocks were prepared in DMSO unless otherwisespecified and are diluted with tissue culture medium to the desiredhigh-test concentration. For each assay, the compounds were then furtherdiluted in tissue culture medium as required. After incubation, thecells were processed to derive, where applicable, EC50 and EC90(compound concentration reducing replicon replication by 50% and 90%respectively). CC50 (concentration decreasing cell viability by 50%) andSI50 (CC50/EC50) values were determined and reported. Anti-HCV activitywas assessed with the replicon (genotype 1b or 2a) or HCVccvirus-derived Luc activity as readout; whereas the cytotoxicconcentrations of drug reducing cell numbers was assessed by theCytoTox-1 cell proliferation assay (Promega, Madison, Wis.) according tomanufacturer's protocol. Recombinant interferon alpha was used as thepositive control drug to validate assay performance.

Assays for Influenza, Respiratory syncytial virus (RSV), and SARS CoV:

Principal Viruses and Cells used were Influenza StrainA/California/7/2009 (H1N1) in MDCK cells, Respiratory syncytial virusStrain A-2 in Hep2 cells and SARS CoV Strain Toronto-2 in VeroE6 cells.

Assays for Influenza virus, RSV, and SARS CoV were Cytopathiceffect/Toxicity-based assay using CellTiter-Glo. The antiviralcytoprotection assays examined the effects of compounds at designateddose-response concentrations in specific cell types to test the efficacyof the compounds in preventing the virus-induced cytopathic effect.Ribavirin was included as a positive control drug for influenza and RSV,while calpain IV inhibitor was used for SARS antiviral assays.Subconfluent cultures of cells were plated into 96-well plates for theanalysis of cell viability (cytotoxicity) and antiviral activity (CPE).For the standard assay, drugs were added to the cells 24 hours later.The CPE wells also received 100 tissue culture infectious doses (100TCID50s) of titered virus. 72 hours later the cell viability wasdetermined.

Measurement of viral-induced CPE was based on quantitation of ATP, anindicator of metabolically active cells. The CPE assay employed acommercially available CellTiter-Glo™ Luminescent Cell Viability Kit(Promega, Madison, Wis.), and was a reliable method for determiningcytotoxicity and cell proliferation in culture. The procedure involvedadding the single reagent (CellTiter-Glo™ Reagent) directly topreviously cultured, subconfluent cells in media. This induced celllysis and the production of a bioluminescent signal (half-life greaterthan 5 hours, depending on the cell type) that was proportional to theamount of ATP present (which is a biomarker for viability).

Assays for Dengue (DENV), West Nile Virus (WNV), Yellow Fever Virus(YFV), Rift Valley Fever Virus (RVFV), Venezuelan Equine EncephalitisVirus (VEEV):

Primary cytopathic effect (CPE) reduction assay. Four-concentration CPEinhibition assays were performed. Confluent or near-confluent cellculture monolayers in 96-well disposable microplates were prepared.Cells were maintained in MEM or DMEM supplemented with FBS as requiredfor each cell line. For antiviral assays the same medium was used butwith FBS reduced to 2% or less and supplemented with 50 μg/mLgentamicin. The test compound was prepared at four log 10 finalconcentrations, usually 0.1, 1.0, 10, and 100 μg/mL or μM. The viruscontrol and cell control wells were on every microplate. In parallel, aknown active drug is tested as a positive control drug using the samemethod as was applied for test compounds. The positive control wastested with each test run. The assay was set up by first removing growthmedia from the 96-well plates of cells. Then the test compound wasapplied in 0.1 mL volume to wells at 2× concentration. Virus, normallyat <100 50% cell culture infectious doses (CCID50) in 0.1 mL volume, wasplaced in those wells designated for virus infection. Medium devoid ofvirus was placed in toxicity control wells and cell control wells. Viruscontrol wells were treated similarly with virus. Plates were incubatedat 37° C. with 5% CO₂ until maximum CPE is observed in virus controlwells. The plates were then stained with 0.011% neutral red forapproximately two hours at 37° C. in a 5% CO₂ incubator. The neutral redmedium was removed by complete aspiration, and the cells may be rinsed1× with phosphate buffered solution (PBS) to remove residual dye. ThePBS was completely removed and the incorporated neutral red is elutedwith 50% Sorensen's citrate buffer/50% ethanol (pH 4.2) for at least 30minutes. Neutral red dye penetrates into living cells, thus, the moreintense the red color, the larger the number of viable cells present inthe wells. The dye content in each well was quantified using a 96-wellspectrophotometer at 540-nm wavelength. The dye content in each set ofwells was converted to a percentage of dye present in untreated controlwells using a Microsoft Excel computer-based spreadsheet. The 50%effective (EC50) concentrations and 50% cytotoxic (CC50) concentrationswere then calculated by linear regression analysis. The quotient of CC50divided by EC50 gives the selectivity index (SI) value.

Assays for Adenovirus (AdV), Measles (MEV), Poliovirus (POV) andEnterovirus (ENTV):

The primary screen was a cytopathic effect (CPE) reduction assay.Briefly, 96-well cultures of cells were infected with virus in thepresence of test compounds and incubated for 4-7 days (depending on thespecific virus/cells). Each virus was pre-titered such that controlwells exhibited approximately 95% loss of cell viability due to virusreplication. Therefore, antiviral effect, or cytoprotection, wasobserved when compounds prevent virus replication. Each assay platecontained cell control wells (cells only), virus control wells (cellsplus virus), compound toxicity control wells (cells plus compound only),compound colorimetric control wells (compound only, no cells or virus),as well as experimental wells (compound plus cells plus virus).Cytoprotection and compound cytotoxicity were assessed by MTS(CellTiter®96 Reagent, Promega, Madison Wis.) dye reduction. The percentreduction in viral CPE (antiviral activity) and percent cell viability(cytotoxicity) were determined and reported.

Assays for Vaccinia Virus (VACV):

The primary assay was a cytopathic effect (CPE) reduction assay. Lowpassage (3-10) HFF cells were trypsinized, counted, and seeded into 96well tissue culture plates in 0.1 mL of MEM supplemented with 10% FBS.The cells were then incubated for 24 h at 37° C. The media was thenremoved and 100 μL of MEM containing 2% FBS was added to all but thefirst row. In the first row, 125 μL of media containing the experimentaldrug (i.e., Compound 1) was added in triplicate wells. Media alone wasadded to both cell and virus control wells. The drug in the first row ofwells was then diluted serially 1:5 throughout the remaining wells. Theplates were then incubated for 60 minutes and 100 μL of a virussuspension was added to each well, excluding cell control wells whichreceived 100 μL of MEM. The plates were then incubated at 37° C. in aCO₂ incubator for three days for VACV. After the incubation period,media was aspirated and the cells stained with crystal violet informalin for 4h. The stain was then removed and the plates were rinseduntil all excess stain was removed. The plates were allowed to dry for24 h and the amount of CPE in each row determined using a BioTekMultiplate Autoreader. EC₅₀ and CC₅₀ values were determined by comparingdrug treated and untreated cells using a computer program.

Example 2—Determination of Efficacy of Compound 1 Against MurineNorovirus in Mice

Methodology

Two studies (Study No. 1 and Study No. 2) examined the ability ofCompound 1 to protect mice from or to reduce murine norovirus infection:

Study No. 1 evaluated the efficacy of twice-daily dosing of Compound 1over a range of 30 mg/kg to 300 mg/kg, initiated prior to infection ofmice with 10⁶ plaque-forming units (PFU) of murine norovirus (MNV) CR3.A control group of mice treated with vehicle was also included. Alldoses were started 2 days (39 hours) prior to infection. Study groupsare shown in Table 3.

Compound was delivered twice daily at indicated doses via oral gavage. Acontrol group of mice treated with vehicle only was also included. Micewere infected with murine norovirus by pipetting virus into mouth 2 daysafter the first dose. The mice used were ˜20 gram, 8-12 week old femaleBALB/c mice in groups of 5 (the study groups are shown in Table 3). Micewere housed on metal grates and combined fecal output was collectedevery 24 hours. Tissues (distal ileum and cecum) and individual fecalpellets were harvested on day 3 post inoculation. All samples weretitered by plaque assay.

TABLE 3 Study No. 1: Study Design for Proof-of-Concept EfficacyEvaluation of Compound 1 against Murine Norovirus Infection in MiceGroup⁽¹⁾ Dosage and Readouts after norovirus (n = 25) Test ArticleDelivery challenge 1 Compound 30 mg/kg/day Pooled fecal samples 1 (BID)orally shed over 24 hour period, starting on day - 2⁽²⁾ analyzed forviral 2 Compound 100 mg/kg/day content via plaque assay 1 (BID) orallyTissue titers and individual starting on day - 2 fecal titers determined3 3 Compound 300 mg/kg/day days post inoculation via 1 (BID) orallyplaque assay starting day - 2 4 Oral vehicle N/A ⁽¹⁾5 mice per group⁽²⁾Compound was administered 51, 39, 27, 15 and 3 hours beforeinfection, and then every 12 hours thereafter Note: 30 mg/kg/day (15mg/kg/dose, bid); 100 mg/kg/day (50 mg/kg/dose, bid); 300 mg/kg/dose,(150 mg/kg/dose, bid). Start oral gave of drug 2 days before injection;BID oral gavage drug.

Study No. 2 evaluated the efficacy of twice-daily dosing of Compound 1at 150 mg/kg or 300 mg/kg, initiated prior to infection of mice with 10⁴pfu of MNV CR3. A control group of mice treated with vehicle was alsoincluded. The 150 mg/kg dose was started either 2 days (39 hours) priorto inoculation, 1 day (15 hours) prior to inoculation, or on the day of(3 hours prior to) inoculation; the 300 mg/kg dose was started 2 days(39 hours) prior to infection. Study groups are shown in Table 4.

This study tested the ability of Compound 1 to protect mice from orreduce murine norovirus infection. Compound was delivered twice daily atindicated doses via oral gavage starting 2 days prior to inoculation, 1day prior to inoculation or at time of inoculation. A control group ofmice treated with vehicle only was also included. Mice were infectedwith 10⁴ PFU of murine norovirus by pipetting virus into mouth 3 hoursafter the Day 0 dose. The mice used were ˜20 gram, 8-12 week old femaleBALB/c mice in groups of 5 (the study groups are shown in Table 4). Allsamples were titered by plaque assay.

TABLE 4 Study No. 2: Study Design for Efficacy Evaluation of Compound 1against Murine Norovirus Infection in Mice Group⁽¹⁾ Test Dosage andReadouts after (n = 25) Article Delivery norovirus challenge 1 Compound150 mg/kg twice Pooled fecal samples 1 daily orally shed over 24 hourstarting on day - 2⁽²⁾ period, analyzed for 2 Compound 150 mg/kg twiceviral content via 1 daily orally plaque assay starting on day - 1⁽³⁾Tissue titers and 3 Compound 150 mg/kg twice individual fecal titers 1daily orally determined 3 days starting day 0⁽⁴⁾ post inoculation 4Compound 300 mg/kg twice via plaque assay 1 daily orally starting day -2⁽²⁾ 5 Oral vehicle N/A ⁽¹⁾5 mice per group ⁽²⁾Compound was administered51, 39, 27, 15 and 3 hours before infection, and then every 12 hoursthereafter ⁽³⁾Compound was administered 27, 15, and 3 hours prior toinfection, and then every 12 hours thereafter ⁽⁴⁾Compound wasadministered 3 hours prior to infection, and then every 12 hoursthereafter Note: 150 mg/kg twice daily = 300 mg/kg total daily dose; 300mg/kg twice daily = 600 mg/kg daily dose. Oragl gave drug for 2 days or1 day or day 0 before infection. Infect mice with 10⁴ PFU MNV via oralgavage on day 0. Twice daily oral gavage every 12 hours.

For both studies, compound was delivered via oral gavage at theindicated doses and times prior to infection. Mice were infected withmurine norovirus CR₃ by pipetting virus into mouth 3 hours after thefirst day 0 dose of Compound 1 was given. Following infection, compoundwas administered twice daily through day 3 post-infection.

The mice used were ˜20 gram, 8-12 week old female BALB/c mice in groupsof 5. Mice were housed on metal grates and combined fecal output wascollected every 24 hours, starting at day −1. Tissues (˜1 cm of distalileum and cecum) and individual fecal pellets were harvested on day 3post-infection and weighed. All samples were titered by plaque assay(qRT-PCR as back-up when titers are too low); titers were normalized togram of tissue or feces. On day 3, duplicate samples of tissue (˜1 cm ofdistal ileum and cecum) were collected, rinsed in PBS, and snap frozen;serum and a duplicate feces sample were also collected on day 3 and snapfrozen; this set of sample was submitted for MS analysis to assess drugbioavailability.

Results

Results of Study No. 1 show that twice-daily Compound 1 treatment,administered starting 2 days prior to infection, was effective inreducing the titer of murine norovirus in both tissue and feces, with agreater reduction in virus titer observed with increasing drugconcentration as shown in FIG. 1A and FIG. 1B. The data also demonstratea significant reduction in virus titer for animals treated twice-dailywith 300 mg/kg Compound 1 in both tissue and feces compared withvehicle.

As shown in FIGS. 1A and 1B, mice were treated via oral gavage with theindicated doses of Compound 1 given twice daily, starting 2 days priorto infection with 10⁶ pfu of murine norovirus as set forth in “Study No.1.”

Results of Study No. 2 demonstrate that treatment of mice twice dailywith 300 mg/kg of Compound 1, starting 2 days prior to infection,significantly reduces murine norovirus titer both in tissue and feces asshown in FIG. 2A and FIG. 2B. These data confirm the findings of Study#1 and demonstrate that Compound 1 is efficacious in reducing murinenorovirus infection.

As shown in FIGS. 2A and 2B, mice were treated via oral gavage with theindicated doses of Compound 1 administered twice daily, starting at theindicated days prior to infection with 10⁴ PFU of murine norovirus asset forth in “Study No. 1.”.

FIGS. 3A and 3B show the number of plaque forming units per gram fromStudy 1 on a linear scale instead of a logarithmic scale. As shown inFIG. 3A, compound 1 dosed orally BID (twice per day) starting on day −2before infection (n=5/group) showed a reduction in PFU/gram withincreasing dose. As shown in FIGS. 3A and 3B, compound 1 reduces mousenorovirus in tissues and stool.

Example 3—Norovirus Polymerase Inhibition Assay

Polymerase reactions (10 μL) were conducted for 60 minutes at 37° C.Nucleoside triphosphates (NTPs) were present at 100 μM each, with 0.05μCi α32P-UTP (800 Ci/mmol). Compounds were incubated with the polymerase(Pol) or pro-polymerase (ProPol), with or without viral proteingenome-linked (VPg) in reaction buffer in the absence of NTPs for 10minutes on ice. Reactions were initiated by the addition of NTPs, andterminated by the addition of an equal volume of 2× Tris/Borate/EDTA(TBE) loading dye/buffer (Invitrogen, Inc.). RNA products (100 nt) wereresolved by electrophoresis in 6% TBE-urea gels (Invitrogen, Inc.).Semi-quantitative analysis of RNA products was conducted by exposure ofdried gels to GE Healthcare Phosphor screens, followed by measurement ofrelative band intensities using GelQuant.NET software (BiochemLabSolutions, Inc.). IC₅₀ and IC₉₀ calculations were obtained using linearregression. The results are given below in Table 5. A comparison with2′-C-methylcytidine triphosphate (2′CmeC TP) is given for reference.

TABLE 5 Norovirus Polymerase Assays Compound Polymerase IC₅₀ IC₉₀ 2′CmeC TP Pol 1.71 ± 0.05 4.45 ± 0.28 2′ CmeC TP Pol + VPg 1.70 ± 0.044.07 ± 0.17 2′ CmeC TP ProPol 1.77 ± 0.04 4.71 ± 0.18 2′ CmeC TPProPol + VPg 1.71 ± 0.06 4.23 ± 0.35 Compound 1 TP Pol 3.41 ± 0.14 9.63± 0.78 Compound 1 TP Pol + VPg 3.37 ± 0.10 9.33 ± 0.72 Compound 1 TPProPol 3.35 ± 0.12 9.78 ± 0.60 Compound 1 TP ProPol + VPg 3.01 ± 0.149.64 ± 0.85

Example 4—Conversion of Compound 1 to Triphosphate in RAW Cells

RAW cells were incubated with Compound 1 at the concentrations shown inTable 6a (ng/cell) and in Table 6b (pmol/cell).

TABLE 6a Conversion of Compound 1 to Triphosphate in ng/cellConcentration (Compound 1- Compound (ng/1 × 10⁶ cells) TP)/(Compound 1)1 Conc. Compound 1 Compound 1-TP (ng) 0.5 μM 0.17 3.74 21.8   1 μM 0.135.13 39.4   5 μM 0.65 26.77 41.3  10 μM 1.02 41.78 41.1

TABLE 6b Conversion of Compound 1 to Triphosphate in pmol/cell.Concentration (Compound 1- Compound (pmol/1 × 10⁶ cells)TP)/(Compound 1) 1 Conc. Compound 1 Compound 1-TP (pmol) 0.5 uM 0.536.37 12.0   1 uM 0.40 8.74 21.7   5 uM 2.01 45.61 22.7  10 uM 3.15 71.1822.6

RAW cells were incubated with four different concentrations of Compound1 for 48 hours in T75 flasks at a density of 1.2×10′ cells/flask. Afterthe incubation period, the cells were rinsed twice with cold PBS andcounted. The cell pellet was suspended in 1000 μL of coldmethanol:distilled water (70:30), vortexed, and frozen at −80° C. untiltime of analysis. As shown in Tables 6a and 6b, Compound 1 and Compound1-TP could be detected in RAW cells treated with Compound 1 atconcentrations from 0.5 μM to 10 μM. The concentration of Compound 1-TPwas 12 to 23-fold higher than Compound 1.

Example 5—Efficacy of Compound 1 Against Human Norovirus

The efficacy of compound 1 for inhibiting norovirus was compared to DMSO(as a control), compound 2, and 2′-C-methylcytidine triphosphate (2′CmeCTP). Cells were pretreated with 25 μM of experimental compound for twohours. Virus inoculum was added for two hours, and unbound virus washedoff and fresh media and fresh experimental compound was added.

The experiment was conducted in duplicate and the results are shown inFIG. 4 (first duplicate) and FIG. 5 (second duplicate). FIG. 6 shows anoverlay of the results of the first and second duplicate of theexperiment. As shown in FIGS. 4, 5 and 6, “A” is DMSO, “B” is Compound2, “C” is 2′-C-methylcytidine triphosphate (2′CmeC TP) and “D” isCompound 1. FIGS. 4-6 demonstrate that the viral titer increased almosttwo orders of magnitude when treated with only DMSO or 2′CmeC TP.However, the viral titer increased less than one order of magnitude inthe presence of compound 1.

Example 6—Effective Concentration of Compounds of the Disclosure andAnalogs Thereof

Table 7 below shows the EC₅₀ and CC₅₀ values of some compounds of thedisclosure as well as analogs thereof for murine norovirus. In caseswhere the compounds were assayed but no EC₅₀ value could be measured,the EC₅₀ value is given as N/A.

TABLE 7 EC₅₀ values for Compounds of Formula II Compound No. EC₅₀ (μM) 11.7-3.6  2 1.9-4.15 3 2.0 4 3.5 5 2.2 6 3.08 71 3.15 77 4.4 76 16.4 10718.1 111 21 126 33.6 38.9 133 25.7 137 20 139 2.3 141 3.2 143 26.3 1457.4

TABLE 8 EC₅₀ values for of Compounds of the Disclosure and Analogsthereof Compound No. EC₅₀ (μM) CC₅₀ 7 53.8/38.4 8 23.6 10 61.511 >121 >121 12 N/A 14 >100 16 >100 17 >100 18 >100 19 >100 20 >20  >20 21 >100 22 >100 27 >100 28 >100 29 >100 30 >100 31 >100 32 >100 33 >10034 >100 35 >100 36 >100 37 >100 38 >100 39 >100 41 >100 42 >100 43 >10044 >100 45 >100 46 >100 47 >100 48 >100 49 >100 50 >100 51 >100 54 >10061 >100 63 >100 65 >100 66 20.2 71.8 68 >100 69 >100 72 >100 73 >10078 >100 81 >100 82 >100 83 >100 84 >100 85 >100 86 >100 88 >100 91 >10093 >100 94 >100 95 >100 96 NA 98 >100 100 >100 103 >100 104 >100106 >100 108 >100 110 >100 114 >100 115 >100 116 >100 117 >100 120 >100121 >100 122 >100 123 >100 124 >100 125 >100 128 >100 129 88.7 130 42.7131 >100 132 >100 134 68.4 136 64.5 138 >100 140 >100 142 >100 144 >1009 50 13 >200 >200 15 >100 >100 23 >100 24 >20  >20  25 >100 26 >10040 >100 52 >100 53 >100 55 N/A 56 75.3 57 >100 58 >100 59 >100 60 N/A62 >100 64 >100 67 >100 70 >100 74 >100 75 >100 79 >100 80 >100 87 >10089 >100 90 >100 92 >100 97 >100 99 >100 101 >100 102 >100 105 >100109 >100 112 >100 113 >100 118 >100 119 >100 135 >100 127 5.5

Example 7—Synthesis of Compound 1

Step 1 (Protocol #1):

To a 100-L jacketed reactor were charged4-amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile(3.00 kg),(3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate (6.60 kg) and DCE (18.89 kg). Stirring was started and DBU(3.61) kg was added. Over a period of 03 h and 14 min, TMSOTf (8.01 kg)was added between 30.6° C. and 37.3° C. IPC after 01 h and 30 min atapprox. 32° C. showed 4% of4-amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile(3.00 kg),(3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate remaining. IPC after 03h and 16 min at approx. 32° C. showed2% 4-amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile(3.00 kg),(3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate remaining (spec: ≦3%). The reaction mixture was diluted withDCM (39.81 kg) and quenched with potable water (15.02 kg) over an 11 minperiod between 9.5° C. and 15.6° C. The extractive work-up (at approx.22° C.) was completed by a back extraction of the aqueous phase with DCM(19.90 kg), a wash with sat NaHCO₃ (1.3 kg NaHCO₃ in 14.9 kg potablewater), a back extraction of the bicarbonate phase with DCM (19.71 kg)and a wash with brine (4.5 kg NaCl in 14.9 kg potable water). Note: thereactor was cleaned with potable water, acetone and DCM after eachwash/back extraction.

The drummed organic phase containing the product was charged to the100-L jacketed reactor through an in-line filter followed by a DCM rinseof the drum and filter with DCM (2.48 kg). The contents of the reactorwere distilled to 31 L with the aid of vacuum over a period of 06 h and04 min with a maximum temperature of 50.1° C. At this point a thicksuspension had formed. Next, over a period of 39 min, IPAc (41.88 kg)was added between 44.5° C. and 49.5° C. and the contents of the reactorwere heated to 76.9° C. over a period of 01 h and 25 min. Next, thecontents of the reactor were cooled to 9.9° C. over a period of 04 h and21 min and stirred for 12 h and 26 min with a minimum temperature of1.6° C.

Step 1 (Protocol #2): To a 100-L jacketed reactor were charged4-amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile(3.00 kg),(3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate (6.60 kg) and DCE (18.80 kg). Stirring was started and DBU(3.59) kg was added. Over a period of 01 h and 46 min, TMSOTf (7.90 kg)was added between 30.4° C. and 34.2° C. IPC after 02 h and 49 min atapprox. 34° C. showed 1% of4-amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrileremaining (spec: ≦3%). The reaction mixture was diluted with DCM (40/70kg) and quenched with potable water (14.97 kg) over an 04 min periodbetween 9.9° C. and 18.0° C. The extractive work-up (at approx. 22° C.)was completed by a back extraction of the aqueous phase with DCM (20.34kg), a wash with sat NaHCO₃ (1.30 kg NaHCO₃ in 14.90 kg potable water),a back extraction of the bicarbonate phase with DCM (20.65 kg) and awash with brine (4.50 kg NaCl in 14.96 kg potable water). Note: thereactor was cleaned with potable water, acetone and DCM after eachwash/back extraction.

The drummed organic phase containing the product was charged to the100-L jacketed reactor through an in-line filter followed by a DCM rinseof the drum and filter with DCM (1.49 kg). The contents of the reactorwere distilled to with the aid of vacuum over a period of 04 h and 49min with a maximum temperature of 45.6° C. At this point a thicksuspension had formed. Next, over a period of 27 min, IPAc (41.70 kg)was added between 45.6° C. and 48.2° C. and the contents of the reactorwere heated to 75.7° C. over a period of 01 h and 20 min. Next, thecontents of the reactor were cooled to 9.4° C. over a period of 04 h and15 min and stirred overnight with a minimum temperature of 2.3° C.

Step 2:

To the reactor were charged(2R,3R,4R,5R)-2-(4-amino-6-bromo-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate (10.0 kg), 10% Pd on C (Degussa, Type E101NE/W),trimethylamine (7.3 kg) and THF (44.5 kg). Hydrogen was submitted to thereactor and the mixture was stirred for 03 h and 54 min between 24.7° C.and 19.6° C. at approx. 30.8 psig. IPC (HPLC) showed that(2R,3R,4R,5R)-2-(4-amino-6-bromo-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate could no longer be detected.

The reaction mixture was filtered over Celite (7.2 kg) and a polishfilter and the filter residue was washed with THF (5.2 kg). The combinedfiltrate and wash was transferred to a 100-L jacketed reactor with theaid of a THF wash (2.12 kg). The contents of the reactor were vacuumdistilled with a maximum batch temperature of 30.0° C. over a period of05 h and 38 min to a final volume of 27 L. IPA (31.48 kg) was chargedover a 40 min period to the reactor between 39.7° C. and 53.2° C. Thecontents of the reactor were vacuum distilled with a maximum batchtemperature of 53.2° C. over a period of 03 h and 02 min to a finalvolume of 33 L. IPA (48.99 kg) was charged over a 43 min period to thereactor between 53.1° C. and 57.1° C. The contents of the reactor wereheated to 60.2° C., agitated for 12 min and cooled over a period of 04and 28 min to 5.4° C. Cold stirring was continued for a period of 08 hand 55 min with a minimum temperature of 1.1° C. The slurry was filteredand washed with IPA (9.41 kg, at approx. 4.5° C.). The residue was driedunder vacuum with a nitrogen bleed for a period of 11 h and 44 min at amaximum temperature of 44.0° C. to provide an LOD of 0.36%. Yield: 6.58kg (73.9%). ¹H NMR confirms structure. Purity: 97.78% (HPLC, AUC).

Step 3:

Materials MW Eqs/vol Amount mmoles Lot # (2R,3R,4R,5R)-2- 617.607 1366.9 594.1 (4-amino-5-cyano- 2-methy1-7H-pyrrolo[2,3-d]pyrimidin-7-yl)- 5-((benzoyloxy)methyl) tetrahydrofuran- 3,4-diyldibenzoate 2.5M NaOH¹ 2.5 910 mL Sigma Aldrich/ 221465/ ACS/ MKBT1665VWater As Potable needed THF 2.5 910 mL Sigma Aldrich/ 360589/ ACS/SHBG3052V 3M HCl² As Sigma needed Aldrich/ 258148 / ACS, 37%/ SHBG3175V¹100 g NaOH dissolved in potable water to a total volume of 1 L;²Diluted 500 mL conc. HC1 in 2 L total with potable water

A solution of(2R,3R,4R,5R)-2-(4-amino-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate and THF was heated to 54° C. and the addition of 2.5 M NaOHwas started. The initial addition gave a biphasic mixture andendothermic response (the temperature dropped to 50° C.) but as theaddition continued a single phased, clear solution formed which wasaccompanied by a fast exotherm to 61° C.; the reaction temperature wasmaintained at 60° C. to 61° C. during the rest of the addition and foran additional 2½ h. IPC showed that no(2R,3R,4R,5R)-2-(4-amino-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate was left.

The reaction mixture was cooled to 21° C. and neutralized with 3 N HClwith external cooling to pH=7.06 (Denver Instrument UB-10 pH meterequipped with a Sartorius P-P11 pH electrode, the electrode was checkedwith buffer solutions of pH=4.00 and pH=7.00); the mixture continued tocool to 8° C. The resulting neutralized mixture was distilled undervacuum with a pot temperature of 45° C. to 50° C. until the emergence ofsolids were observed in the pot. The suspension was cooled and stirredfor 2 h at 2° C. The beige suspension was filtered to afford a darkfiltrate; the off-white residue was washed once with cold water (500 mL,5° C.). A first LOD after 16 h gave a value of 18.73%. HPLC) of thedrying material showed the presence of 1.6% benzoate.

A brief rework study for compound 1, (containing 1.6% benzoic acid perAUC, HPLC) was executed in 10 vol of water (1 g in 10 mL):

-   -   3 h slurry at ambient    -   3h slurry at 50° C.    -   24 h slurry at ambient

All three experiments gave compound 1 with less than 0.1% benzoic acid(UAC, HPLC). The slurries were fluid, were easily stirred and filtrationwas fast. Short term drying on the filter gave a powder-like solidindicating that a displacement wash with an organic solvent is notneeded. Without wishing to be bound by theory, a loss of NMT than 1% isexpected (solubility 1 mg/mL). HPLC data for compound 1 were obtainedwith a method suitable for polar compounds using a Zorbax Eclipse PlusC18 column (water/ACN/TFA, 97.5/2.5/0.05). This is the same column usedfor steps 1 and 2.

The cold product suspension was filtered and the reactor and residuewere washed with cold IPAc (approx. 7.5° C., 13.16 kg and 13.62 kg)until a colorless filtrate had been obtained. The residue was driedunder vacuum and a nitrogen bleed ≦45° C. for a period of 65 h and 19min to an LOD of 0%. Yield: 5.87 kg (70.7%), ¹HMNR confirmed identity;HPLC purity 98.84% (AUC).

EQUIVALENTS

The disclosure can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the disclosure described herein. Scope of thedisclosure is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A compound of Formula II:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof, wherein: Y is —C(O)—, or

wherein X¹ is independently O, NH, or S, X² is independently a bond,—O—, —S—, or —NH—, and X³ is independently —OR, —NHR^(II), or —SR^(II);each R is independently —H, —C₁-C₂₀alkyl, —C₂-C₂₀alkenyl,—C₂-C₂₀alkynyl, —C₃-C₈ cycloalkyl, —C₄-C₈ cycloalkenyl, aryl,heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl isoptionally substituted with one or more halogen, oxo, R¹, —OR¹, —NR¹R²,—SR¹, —OC(O)R¹, —C(O)OR¹, —NHC(O)OR¹, or —NHC(O)R¹; R^(a) and R^(b) areeach independently, at each occurrence, —H, —C₁-C₂₀alkyl,—C₂-C₂₀alkenyl, —C₂-C₂₀ alkynyl, —C₃-C₈ cycloalkyl, —C₄-C₈ cycloalkenyl,aryl, heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl isoptionally substituted with one or more halogen, oxo —OR¹, —NR¹R², —SR¹,—OC(O)R¹, —C(O)OR¹—NHC(O)OR¹, or —NHC(O)R¹; R¹ and R² are eachindependently, at each occurrence, —H, —C₁-C₂₀alkyl, —C₂-C₂₀alkenyl,—C₂-C₂₀alkynyl, —C₃-C₈ cycloalkyl, —C₄-C₈ cycloalkenyl, aryl,heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl isoptionally substituted with one or more halogen, oxo, —R³, —R⁴, —OR³,—NR³R⁴, —SR³, —OC(O)R³, —C(O)OR³, —NHC(O)OR³, or —NHC(O)R³; R³ and R⁴are each independently, at each occurrence, —H, —C₁-C₂₀alkyl,—C₂-C₂₀alkenyl, —C₂-C₂₀alkynyl, —C₃-C₈ cycloalkyl, —C₄-C₈ cycloalkenyl,aryl, heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl isoptionally substituted with one or more halogen, oxo, aryl, heteroaryl,—OH, —NH₂, —SH, —OC(O)H, —C(O)OH, —NHC(O)OH, or —NHC(O)H; R^(c) isindependently —H or -D; and n is independently 0, 1, 2 or
 3. 2. Thecompound of claim 1, wherein R^(a) is —H.
 3. The compound of claim 1,wherein R^(b) is —H.
 4. The compound of claim 1, wherein R^(c) is —H. 5.The compound of claim 1, wherein n is
 0. 6. The compound of claim 1,wherein R^(a), R^(b) and R^(c) are —H.
 7. The compound of claim 1,wherein R^(a), R^(b) and R^(c) are —H and n is
 0. 8. The compound ofclaim 1, wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 9. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 10. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 11. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 12. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 13. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 14. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 15. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 16. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 17. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 18. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 19. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 20. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 21. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 22. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 23. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 24. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 25. The compound of claim 1,wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 26. A pharmaceuticalcomposition comprising a compound of claim 1, or a pharmaceuticallyacceptable salt, solvate, enantiomer, diastereomer, racemate or mixturethereof, and a pharmaceutically acceptable carrier.
 27. Thepharmaceutical composition of claim 26, wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof, and a pharmaceuticallyacceptable carrier.
 28. A method of treating a viral infection or aviral-infection associated disease or disorder comprising administeringto a subject in need thereof an effective amount of a compound of claim1, or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 29. A method of treating aviral infection or a viral-infection associated disease or disordercomprising administering to a subject in need thereof an effectiveamount of the compound:

or a pharmaceutically acceptable salt, solvate, enantiomer,diastereomer, racemate or mixture thereof.
 30. The method of claim 29wherein the viral infection is norovirus.